This article addresses the potential clinical value of techniques based on surface electromyography (sEMG) in rehabilitation medicine with specific focus on neurorehabilitation. Applications in exercise and sport pathophysiology, in movement analysis, in ergonomics and occupational medicine, and in a number of related fields are also considered. The contrast between the extensive scientific literature in these fields and the limited clinical applications is discussed. The “barriers” between research findings and their application are very broad, and are longstanding, cultural, educational, and technical. Cultural barriers relate to the general acceptance and use of the concept of objective measurement in a clinical setting and its role in promoting Evidence Based Medicine. Wide differences between countries exist in appropriate training in the use of such quantitative measurements in general, and in electrical measurements in particular. These differences are manifest in training programs, in degrees granted, and in academic/research career opportunities. Educational barriers are related to the background in mathematics and physics for rehabilitation clinicians, leading to insufficient basic concepts of signal interpretation, as well as to the lack of a common language with rehabilitation engineers. Technical barriers are being overcome progressively, but progress is still impacted by the lack of user-friendly equipment, insufficient market demand, gadget-like devices, relatively high equipment price and a pervasive lack of interest by manufacturers. Despite the recommendations provided by the 20-year old EU project on “Surface EMG for Non-Invasive Assessment of Muscles (SENIAM),” real international standards are still missing and there is minimal international pressure for developing and applying such standards. The need for change in training and teaching is increasingly felt in the academic world, but is much less perceived in the health delivery system and clinical environments. The rapid technological progress in the fields of sensor and measurement technology (including sEMG), assistive devices, and robotic rehabilitation, has not been driven by clinical demands. Our assertion is that the most important and urgent interventions concern enhanced education, more effective technology transfer, and increased academic opportunities for physiotherapists, occupational therapists, and kinesiologists. Prevention of injuries and rehabilitation of movement pathologies are among the branches of clinical practice where the impact of technology is leading to improvement of outcomes and to economic benefits. In times of limited resources, the future perspectives and developments of all rehabilitation-related professions will increasingly depend on the evidence supporting the effectiveness of their preventive and therapeutic interventions. This issue has been discussed in a number of scientific contributions and editorials –3.In the last few decades, impressive developments have taken place in many fields providing powerful quantitative approaches toward instrumentation-based assessments in cardiology , neurology , and biomechanics . Neuroengineering has made available a wealth of investigational techniques and tools, as well as tutorials and textbooks, for understanding mechanisms, implementing prevention, and measuring performance and results of interventions. The proceedings of the International Conferences on Neurorehabilitation –8 providAs a “muscle activation measuring tool” sEMG has played a growing and important role in neurorehabilitation over four decades –19. FiguDespite this large body of knowledge, literature, and collected research works, the clinical acceptance of sEMG advances among physiotherapists (PTs), kinesiologists and medical clinicians remains low . This isA number of “barriers” exist limiting the widespread application of sEMG techniques in clinical assessment and in neurorehabilitation. Some barriers are cultural, such as the inappropriate comparison with the diagnostic power of needle EMG , 36, or It is the objective of this work to discuss these barriers and the options for reducing them.Surface EMG can be used in monitoring neuromuscular pathologies, in prevention of work-related disorders and occupational therapy, and in monitoring neuromuscular changes/progress in acute patients (see section Surface EMG Applications). Information on muscle activation during a movement or effort adds to the clinical evaluation and provides a picture of both impairment and functional alteration. As in other branches of medicine, clinical assessment does not always provide the information needed to design a treatment plan. It is crucial to recognize situations in which added value can be expected from instrumental analysis. Some example of questions which can be answered with sEMG, and which have an impact on designing a rehabilitation plan, are presented in section Some Fundamental Questions. Measurement of muscle activation provides information on the motor unit recruitment/derecruitment capability, on fatigue, synergies, co-contractions, etc. as well as evidence of the efficacy of the rehabilitation plan. Section Surface EMG Applications provides examples of applications.Physiological and technological literacy is a requirement for medical and health-allied professionals.The measurement of a physiological quantity  is useless if the recipient does not know its meaning, how to use the information contained in the result and how reliable the measurement is. An ECG does not convey much information to a person knowing little about cardiac electrophysiology. Similarly, information about muscle fiber conduction velocity, sEMG patterns, and amplitude or frequency spectrum, etc. do not inform a person knowing little about muscle electrophysiology and basic signal analysis. The insufficient competence of instrument operators in performing and interpreting such measurements leads to the predictable conclusion that measurements do not contain clinically useful information.In many countries, the lack of this literacy in physiology/technology leads to the education of physiotherapy and occupational therapy graduates, and future teachers, who serve primarily as professional operators with empirical knowledge mainly . This isThe most important bioelectric signals are generated by the heart (ECG), the brain (EEG), and the muscles (EMG). The technologies for collecting, reading and interpreting these signals were developed 50–60 years ago and reported in many review papers and books –34, 43. Surface EMG is more than a century old. It started with the pioneer work of Piper, Kugelberg, and Denny-Brown –46 and pThe sEMG signal is the algebraic sum of the motor unit action potentials (MUAP) generated by the active motor units (MU) and detected over the skin. Like any other signal, sEMG provides quantitative information concerning wave-shape, amplitude, power spectral density, etc. Using such information or those derived by visual observation is a clinical choice/decision.Traditionally, this signal is detected between two electrodes aligned in the direction of the fibers . This “conventional” bipolar sEMG provides ready answers to many important questions in rehabilitation. It is simple to apply on multiple muscles, even by non-technical users, and provides reliable information on the general activation of a muscle or muscle groups and on temporal events of muscular activation . ConventAdvanced sEMG technologies provide a much larger amount of physiological information than the simple bipolar technique. For example, the use of surface electrode arrays, enables the detection of a so called sEMG “image” that is evolving in time like a movie on the skin [see examples in ]. This tThe current applications of sEMG mostly concern physiological investigations, monitoring of neurological disorders, planning of treatments, assessment of interventions and control of prostheses and robots (section Surface EMG Applications). For reasons of space, only a few references are provided here for each area of application. A much more extensive description of applications is provided in chapters 10–20 of Merletti and Farina .Like other bioelectric signals, sEMG provides a fundamental added value to the assessment of the organ generating it. The information about muscle activation has different forms  and is relevant in many fields ranging from orthopedics and neurorehabilitation, to movement analysis in exercise and sport, from aging to gnathology, from obstetrics to occupational and space medicine , 54–60. Most of the available literature on sEMG concerns methodological issues and proof of concepts, carried out mostly on healthy subjects. Clinical works on large patient groups are few, as well as case studies and case-series on small samples. This does not mean that the developed techniques have no clinical applications or do not answer clinical questions. Rather, it means that there is a huge gap in translating techniques to the clinical environment . Some articles question the diagnostic and therapeutic value of sEMG , 36, 63.The availability of evidence to support rehabilitation has been a long-standing challenge. The complexity of designing clinical trials to assess the efficacy of rehabilitative treatments has become a topic in the current literature . Since sThe sEMG signal is affected by a large number of factors reflecting the pathophysiology of the muscle and of its control strategy. As such, it provides a window into the muscle, the peripheral (PNS), and central nervous system (CNS). These factors range from alteration of the MUAP propagation along the motor unit fibers to the control of force by recruitment/derecruitment and firing rate of the individual motor units. Unraveling the large amount of information contained in the signal is a major technological challenge and an important field of current engineering, physiological and clinical research , 61, 62.Basic and clinical neurophysiology are fields in which sEMG has been extensively applied , 74–77. It was demonstrated early on that sEMG is suitable for the detection of co-activation of agonist and antagonist muscles, whereby physiological activation patterns could be distinguished from pathological ones –80. The Muscle activation patterns, represented by the sEMG envelopes of a few muscles, can be decomposed into a limited number of “basic” functions or patterns, called “synergies” or “primitives.” These primitive patterns can be combined, with different individual weights, and result in the apparent modular organization of multi-muscle activities across different motor tasks. It has been proposed that the nervous system simplifies muscle control through such modularity, using these basic synergies (primitives) to activate muscles in groups. This discovery has had a huge impact on the analysis of motor control and neurorehabilitation since it implies that the CNS generates forces and movements by optimizing the control strategy of either individual muscles or (more likely) muscle synergies –101. ResThe net torque at a joint is usually produced by a number of muscles, ligaments and other passive structures, and by external forces . The force contributed by each individual muscle may fluctuate (as does the sEMG amplitude of the muscle) while the total measured torque may remain constant. The estimation of force sharing among synergic muscles by means of sEMG has been reviewed by Perry 30 years ago , and morThe term “muscle fatigue” has many definitions mostly associated with measurements performed during an isometric constant force contraction which is a common and important “bench-test” condition. One definition considers mechanical fatigue as the inability to sustain a given contraction level and is associated with the endurance time (in isometric constant force contractions) or to the inability to perform a task. Another definition refers to “myoelectric manifestations of muscle fatigue” and considers fatigue as the set of changes affecting sEMG features from the very beginning of the contraction. Its main indicator is muscle fiber conduction velocity (CV) which decreases more or less rapidly depending on the level of contraction and is usually measured during isometric constant force contraction. Both types of “fatigue” depend on blood flow and on the stability of the recruited MU pool. Blood flow is blocked, and all MUs are recruited, at contraction levels above about 50% MVC in most muscles , 106–110The first use of electrode arrays was described by Gydikov in 1972 , 52, 112A textbook is available with the location of the IZs of 43 muscles . KnowledNeuromuscular electrical stimulation involves the application of electrical stimuli to a nerve, or to the motor point of a superficial skeletal muscle, with the objective of inducing and controlling muscle contractions. The stimulus strength (either current or voltage or pulse width) determines the number of recruited motor units whereas the stimulus frequency determines their synchronized discharge rate. Since all the recruited MUs are activated synchronously, effectively as a single large MU, the sEMG signal is deterministic rather than stochastic and is referred to as M-wave or Compound Motor Action Potential (CMAP). Confounding factors and the effects of variability of CNS control, present in voluntary contractions, are eliminated and myoelectric manifestations of muscle fatigue are easy to measure. The technique provides a powerful bench-test for the quantitative investigation of a muscle's electrical and mechanical properties –128. FinThe most common definition of spasticity goes back to Lance, according to whom spasticity is “…a motor disorder, characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerks, resulting from hyper-excitability of the stretch reflex as one component of the upper motor neuron syndrome” and does “not include impaired voluntary movement and an abnormal posture” . Beside Many different studies have shown that sEMG can quantify alterations associated with spasticity, for example through the extraction of sEMG primitive synergies . HoweverFollowing lesions to the CNS, such as stroke, traumatic brain injury, etc., patients can develop acquired deformities at the lower limb that impair or inhibit walking. These deformities, often termed contractures, are due to a combination of paresis, muscle overactivity, spasticity, along with mechanical barriers, including muscle shortening, increased muscle stiffness and viscosity, and retractions. Other phenomena  have to be assessed in dynamic conditions, because they may not be detectable during the bedside evaluation, and may be present only during walking, or vice versa , 67, 132Next, the recruitment of dorsiflexor muscles during walking, regardless of their voluntary activation during tests at the besides, is useful to further tune the surgical plan  , 148. SiSurface EMG-derived indices are also used as outcome measures to evaluate the responsiveness to treatments . In clinIn conclusion, in patients with either acquired CNS lesions  or degenerative diseases , sEMG can be used (1) to better understand the underlying mechanisms of gait deterioration, especially where multifactorial causes coexists, (2) to support the clinical decision making and/or the rehabilitative pathway, and (3) as a marker of disease progression or intervention effectiveness . The samBased on the pioneering work of Joseph and Nightingale , we now This mechanism is altered by age and many pathologies and sEMG provides means to monitor such alterations. The role of sEMG is of paramount importance in helping investigators understand how neural regulation contributes to the prevention of falling, by studying the control of posture and the responses to postural perturbations in healthy and pathological subject –160.The majority of rehabilitative treatments delivered by physiotherapists are related to orthopedic pathologies. In this field, the available sEMG-based indices assessing muscle activation, symmetry and localized fatigue can be used to support the selection of the therapeutic exercises and to monitor their effectiveness over time . In patiAt the shoulder level, alterations in neuromuscular control of the scapular muscles has been proven in subjects with subacromial pain syndrome based on sEMG data . The effLower limb orthopedic pathologies, related to either sport activity or injuries, have been widely investigated with sEMG-based techniques. For example, sEMG has been used to compare the activation of gluteal muscles between healthy and injured runners, to quantify thigh muscle imbalance in subjects with patellofemoral pain –172, andSurface EMG detected from the residual muscles of an amputee has been used for controlling the motors of arm/hand prostheses (myoelectric prostheses) for five decades. This technique is limited to 2–3 basic commands and movements, is not intuitive and requires that the subject learns to associate a specific muscle contraction to the desired output. Recently the technique based on “Targeted Muscle Reinnervation (TMR)” has been tested with success in subjects with amputation at the shoulder level. The residual nerves from the amputated limb are surgically transferred to other, previously denervated, muscles that are not used by the subject. Reinnervation of these muscles takes place within 3–6 months. For example, branches of the median, radial, musculocutaneous, and ulnar nerves may be grafted to specific regions of the serratus or pectoralis muscles. Once reinnervated, these muscles act as biological amplifiers of the neural commands meant for the missing muscles and their sEMG can be detected with electrode arrays and decoded, by pattern recognition processes, for the control of the motors of the prosthesis. The command is therefore intuitive, that is the amputee attempts to move the missing arm and the mechanical arm moves as “desired.” While some problems need to be solved, this technique is highly promising for specific amputees –178.Pregnancy and high-impact sport activity are considered as risk factors for pelvic floor dysfunctions, including urinary incontinence. Surface EMG data demonstrated significant test-retest reliability and significant clinical predictive validity for urinary stress and urge incontinence. Pelvic floor muscle sEMG is reliable and consistently predictive of several important clinical status variables, it can be a useful tool in early detection and prophylactic intervention for muscle laxity. Recent advances in sEMG technology make it cost-effective, convenient and easy to learn and administer by trained assisting staff. This technology is a powerful complementary tool for digital assessment of pelvic floor muscles and should be considered for use in gynecologic practice. Prenatal exercise programs, supported by pelvic floor muscle exercises, should be recommended for pregnant women, especially those who are accustomed to higher exercise intensity –181. SurHigh density surface EMG (HDsEMG) signals have been used for mapping the activity of the muscles surrounding the vaginal, the urethral and the anal canals , 184. HaAlthough very controversial and discouraged, episiotomy is still a widely performed surgery during child delivery. The techniques described above for the location of MU innervation zones (IZ) provide a tool to estimate the risk of partial denervation of the external anal sphincter (EAS) consequent to episiotomy. An intra-anal probe with a circumferential array of 16 electrodes detects the sEMG activity of the EAS during a voluntary contraction. Proper software identifies the location of the IZs of motor units of the EAS. This information can then be used, at the time of delivery, to guide episiotomy (if necessary) to the right or left side to minimize the risk of EAS partial denervation and possible future incontinence. Surface EMG techniques in ergonomics and occupational medicine for prevention and monitoring of occupational disorders were developed in the 90s , 187 andThe literature concerning sEMG applications in sports is very extensive and focused on physiology , 192 traTemporomandibular disorder (TMD) is a term indicating musculoskeletal disorders in the jaw muscles and/or the temporomandibular joint. Surface EMG analysis has been employed to obtain a better understanding of TMD and is useful to elucidate the masticatory muscle function and adaptation in patients with TMD, using indices of dominance, asymmetry, co-ordination and co-operation of temporalis and masseter muscles during mastication. An association exists linking decreased activity to increased severity and asymmetry between affected and non-affected side in unilateral TMD patients –208. TheThe above list of sEMG application fields is far from comprehensive. Many other fields take advantage of sEMG as a tool for either investigation, clinical assessment or treatment. These fields deal with the joint use of sEMG and ultrasound , the stuSection Surface EMG Applications provided a partial overview of the rapidly growing applications of sEMG in neuro-rehabilitation, movement sciences, occupational and sport medicine, and other fields. Many of the thousands of articles in these fields come from rehabilitation engineering groups, quite a few from movement science laboratories, but relatively few from the clinical world. Despite the extremely large number of publications about signal detection, processing tools, and small clinical studies, publications on routine applications in clinical practice are minimal. In addition, sEMG applications in medium/large clinical studies are rare , 73, 219Hardware and software for sEMG detection and processing/interpretation has been developed mostly by academic researchers whose objective is to develop scientific innovation, and to publish their findings in respected science journals. In these cases, the research questions are typically technical, rather than clinical. The giant step between publishing a method, or developing a prototype, and the design, manufacturing, and marketing of a device for clinical applications is expensive and can be undertaken by companies only if there is evidence of clinical effectiveness and demand from the market. But a demand from the market implies some awareness of the users and their understanding of the potential use and relevance of the new method/device. If the users do not know why measuring muscle activity may be important, they will not be interested in a device or method to measure it. This brings up the issue of information, education and knowledge-transfer, but also other considerations concerning the potential consequences that making such measurement will imply. For example, who is qualified to decide on the measurement, who performs it, and who will use and interpret the resulting information to make decisions? Which balances will be affected, what will the costs or savings be, and who will gain something from doing it? This is important in private health management systems, where the role of insurance companies conditions the market, as well as in national health systems, where the state (that is the community) covers health cost with tax money.Most of the scientific breakthroughs produced in academia do not routinely result in a marketable product or procedure. Commercialization of emerging technological innovations is difficult to accomplish; Transferring technological knowledge can also be a time-consuming process resulting often in a market failure. This may be because users are not yet competent or did not contribute to the development of the knowledge. On the other hand, academia-based researchers may also not be competent to assess the needs and boundaries of the clinical procedures. This scenario has been conceptualized as a “valley of death” between research and market. To tackle this general problem, major challenges are being analyzed, at the EU and national levels, and in the USA, involving educated/informed Communities of Practitioners (CoPs) in the process of effective transfer of high-value emerging technologies .In addition to this, recent EU grants required the participation of companies as partners of funded projects.Is the muscle active or not at a given time? When does the muscle turn on and off during a task?Is the muscle relaxed or active or progressively changing its activation level? What level of force is produced?Is muscle activity triggered by muscle lengthening and/or by the velocity of the stretch?What is the level of muscle activation? Is the estimation of force (or force change) of any interest?How are many muscles coordinated and what are the temporal relations between their activations?Is there co-activation of different muscles during a task?Is there a region of a muscle that is more or less active then other regions? Or is there a muscle of a group that is more or less active than other muscles of the same group?What is the strategy adopted for controlling motor unit recruitment and muscle coordination?Where are the innervations zones of the MUs of a muscle located along the muscle?How long are the muscle fibers and how much are they shortening/lengthening during a task?Is conduction velocity of the muscle fibers relevant for the situation at hand? What is its average value? What is its distribution across motor units normal or not?Is muscle fatigue of interest in the situation at hand?Is the number of active motor units of a muscle stable, or changing in time during a task? Are the active motor units rotating, that is are they being de-recruited and replaced by others during performance of a task?What is the fiber size and fatigability of MUs?To address the barriers to clinical use of sEMG, we believe it is important, together with the CoPs, to try to answer fundamental primary questions and a set of secondary questions which outline the potential added value that can be provided by sEMG-based assessments. Primary questions concern the pathophysiological status of the neuromuscular system as well as the definition/measurement of key characteristic features . Secondary questions concern when and why the answer to a primary question is clinically important, who should answer the question and how this person should be trained. The list below includes a few examples of the many primary questions that can be answered by a competent analysis of sEMG:The list below includes examples of secondary questions that should be asked and answered in association to each of the primary questions listed above:When and why is the answer to this primary question important?What must be measured to answer the primary question? What instrument should be used?What is the knowledge required to answer this primary question?Who is the competent clinical operator who should perform the measurements required to answer this primary question?What is the knowledge that such operator must have in order to perform the measurement? How can this knowledge be acquired by this operator?Who is the competent clinical operator qualified to interpret the results of the measurement and draw conclusions from them? How can the required competence and expertise be acquired?The current limited clinical application of sEMG indicates that either type of questions are rarely asked. However, the literature unquestionably indicates that the answers to the primary questions are available and that they are important for understanding the pathophysiology of muscle conditions and motor control strategies. The issue is whether such questions are considered to be relevant, from the clinical viewpoint, by the CoPs, and which barriers prevent answering the relevant ones.As indicated above and in section Introduction, large studies, and translational efforts from scientific knowledge to clinical application are hindered by a number of barriers a few of which have been previously investigated by other authors and identified as (a) lack of time, (b) lack of skills, (c) misperceptions of EBP , 221–223The cultural barriers limiting the widespread use of sEMG are not specific to this field and are the same that affect many other rehabilitation fields. They are, in general, related to the global approach to measurement in rehabilitation and to the concept of evidence provided by such measurements .In physics, “measurement” is the process of attributing a value to a physical quantity by comparing it to a standard reference quantity called “unit of measurement.” In rehabilitation, “measures” can be measurements of physical quantities , ratings of a specific ability based on an ordinal scale with known levels , ratings of multidimensional abilities on item scores that are summed up to obtain a total score , results of questionnaires or aggregations of tests (pass/fail) or ordinal grades, such as very-poor/poor/sufficient/good/very-good, or 0/1/2/3/4/5 . The exact definition of each level of the scale may change from assessor to assessor. On the one hand, all these tools are useful, easy to be administered and represent a key element for both clinical activities and administrative procedures . Noteworthy, dichotomous variables are the pillar of epidemiological studies . On the other hand, they may suffer from metrical issues, from construct validity to sensitivity or reliability, and may lead to huge data-analysis problems when ordinal scores  are treated as numbers and averaged or analyzed with parametric statistics and when the effects of a treatment are computed as numerical difference between scores obtained before and after the treatment. Moreover, electrophysiological variables, such as those describing motor control, cannot be assessed by clinical scales and require instrumental measurements. Some well-known textbooks, such as “Measurement in Neurological Rehabilitation”  do not eWhile physical measurements are the foundation of science and associate the change of a quantity as an effect due to some cause, the classification of the patient's current status or functional ability is the main goal of the “measures” in rehabilitation. Surface EMG amplitude  and spectral  features or timing during movement or tasks, are measurements of muscles signals that in turn reflect and quantitatively describe pathophysiological events or conditions or recovery level. In some cases, the possibility of turning this numerical information into clinically meaningful categories, based on a-priori knowledge and thresholds, would probably support the use of sEMG based examinations (but not of sEMG itself) in the rehabilitation practice.It has been properly pointed out that the statement “there can be no evidence in rehabilitation” that is so often heard from medical operators who think that their job is more “humanistic” than “scientific,” challenges the scientific basis of rehabilitation . Such thFundamental concepts of mathematics and biomechanics are associated to sEMG measurements and to the need for a language common to clinicians and rehabilitation engineers. Efforts in this direction are under way  , 61, 62.Among rehabilitation professionals, there is a tendency to consider sEMG as a therapeutic tool so that the potential benefits of sEMG appear limited to biofeedback applications. In fact, sEMG is much more a monitoring tool, and occasionally, a diagnostic tool. The incorrect view of sEMG as a “therapy” is a barrier to its use.The clinical interpretation of sEMG is based on the timing, amplitude and the morphology  of the signal. Technical aspects related to the type of electrodes, the type of protocol used, the adopted filters, etc., affect the waveform, timing, amplitude, and spectrum of the signal. Also, the modification of the peripheral properties of the muscle and the modification of the central drive have an effect on the morphology of the sEMG signal . Althoughttps://www.wcpt.org/policy/ps-descriptionPT). The academic programs in movement sciences often provide a more scientific and research oriented background.Reading a sEMG recording and linking a pathophysiological and/or biomechanical meaning to its features (that often result from computer processing) requires considerable competences. These are rarely available in the clinical environment. Educational barriers are a bottleneck. Many countries offer a Master in Health Professions . These degrees too often focus on legal, professional and administrative issues and neglect scientific and technical education. Noteworthy, The World Confederation for Physical Therapy (WCPT) advocates that the scope of physical therapist practice is not limited to direct patient/client care, but also includes research  BS program is barely sufficient to train a practitioner, not a contract professor or a clinical researcher able to promote and conduct large-scale studies. In addition, it is unthinkable that a practitioner will acquire this knowledge on his/her own time, in parallel to a heavy burden of clinical work, and publish in qualified journals to achieve an academic status . The sEMIn countries in which physiotherapy is not a graduate level degree, students are trained to become practitioners rather than clinical researchers. The concept of measuring physical quantities is neglected as well as the fundamentals of physics and biomechanics . Moreover, in countries that do not grant a PhD in physiotherapy or movement sciences, teachers of physiotherapy have in general, no or very limited research exposure or international experience. Insufficient continuing education and involvement of teachers in research projects is a barrier to clinical use of all new technologies, and sEMG in particular.Technological evolution led to the development of sEMG hardware that is simple to use and is commercially available. Powerful software can extract sEMG features whose clinical relevance is documented in the available literature. Nevertheless, there is a persistent demand for engineers to build systems that can be easily applied without a high risk of error. Users demand to be technically supported in the interpretation of signals and warned of potential misuse and acquisition error. There is a high demand for artificial expert systems and explanatory components that should be integrated into the sEMG systems and protect the user from errors and misinterpretations. However, no software will correct basic human errors . This brings up the problem of the degree to which lack of competence can or should be replaced by expert systems, artificial intelligence or automatic devices. This may be a dangerous avenue of research in a field where developers and users have widely different expertise, experience and responsibility. Even if software is subject to the same stringent and reliable regulations as all medical devices, it cannot be fool-proof and cannot replace human expertise and competence. The solution is a more competent operator possibly assisted by a more intelligent machine providing warnings or “suggesting” possible interpretations.Many researchers made remarkable efforts to (a) introduce sEMG as a tool to integrate biomechanical information for movement analysis, and (b) to provide tutorials and guidelines to clinical operators , 226–228The new Medical Device Regulation (MDR) of the European Union requires proof of benefit through clinical studies, based on the Health Technology Assessment (HTA) procedure. This will be difficult to provide for sEMG equipment because of the lack of suitable personnel, creating a vicious circle. In addition, the producers of medical devices will have to maintain competencies in the areas of quality assurance and risk management. This is difficult to ensure, especially for very small companies, and may further hinder the translation of innovative sEMG procedures in the long term. Unresolved reimbursement issues and new regulatory barriers will hinder the development of sEMG systems adapted to clinical needs and their translation into clinical practice.As mentioned by Duncan and Murray  “Whilst In most countries, a physiotherapy session lasts 30–60 min and is usually related to treatment, not to measurement of results. This time constraint is mentioned in many articles reporting results of questionnaires or interviews to physiotherapists , 40. In In addition, these authors indicated that “Despite barriers, participants were eager to learn about sEMG, noting that it would not replace but enhance their current clinical methods….” Due to the lack of training, the request is that sEMG equipment and testing should be easy to self-learn, fast and simple to use and inexpensive. The lack of teaching associated to self-teaching by trial and errors or from salespersons only, causes user frustration and is a major barrier to the use of sEMG. This is not so in the case of ECG and EEG (and needle EMG) whose users are provided with proper academic education and training.Cost of FDA or CE-approved sEMG equipment ranges from about 10 k$ to nearly 40 k$ for wireless systems providing up to 32 channels and processing software. This is in the same order of magnitude of inexpensive to sophisticated ECG, EEG, and needle EMG equipment.A possible explanation for the differences between ECG, EEG, needle EMG, and sEMG is that the former have a higher diagnostic yield while sEMG only provides information on the functional level, which is associated with prevention, monitoring, assessment, and treatment planning but less to diagnosis. The lower importance attributed to the latter functions with respect to diagnosis is a bias that is hard to overcome and has high social and economic costs.A vicious circle exists between the need to collect more evidence of sEMG effectiveness in assessing results, and the lack of qualified clinical researchers able to do it. This is a clinical activity that should not be left to either the manufactures or to the rehabilitation engineers. It is a clinical activity dealing with, and requiring, studies on patients. Despite the large number of publications on small studies, the evidence does not seem to be sufficient to convince insurance companies or National Health Systems to reimburse the cost of sEMG-based testing for effectiveness of treatments. In North America, sEMG procedures are not routinely reimbursed by insurance, unless they are part of a preoperative protocol, such as used for surgical planning in patients with CP. This is in contrast to diagnostic procedures using intramuscular needles which are done routinely by clinicians for diagnostic reasons.Evidence supporting the use of sEMG is only partially available because it is limited to small studies. Large studies require substantial funding and competent operators. Competent operators are lacking because of educational barriers. Educational barriers are lower in countries where post-graduate academic degrees are available. Therefore, large-scale studies should be proposed where researchers are available to implement them. Research funding is required to support researchers and pay for equipment and management of large studies.Hundreds of publications on peer-reviewed journals  provide In this work, the barriers to a widespread use of sEMG have been classified into four main groups: cultural, educational, technical, and economic. These are strictly linked and interdependent.Cultural barriers derive from “uneasiness” with technology, from communication gaps, different perceptions and approaches between rehabilitation engineers and clinical operators. These different perceptions hinder technology transfer and generate educational barriers. Overcoming these barriers requires a strongly interdisciplinary educational approach. The lack of a partially overlapping high-level education, involving rehabilitation professionals and engineers, results in different languages, communication gaps, different approaches to common problems, or, in one word, cultural barriers that delay technology transfer. The development of common languages at common institutions would promote the use of sEMG systems and other measurement techniquesTo this end it is important to point out and underline the series of open access Tutorials , 62 and Overcoming educational barriers requires (a) a greater degree of bidirectional osmosis between the clinical and the research environments, (b) funding of translational efforts, (c) use of textbooks and manuals related to the clinical use of sEMG in specific applications prepared by experts , (d) desThe lack of higher academic degrees in physiotherapy and movement sciences prevents (a) the education of qualified researchers able to properly apply the rapidly developing technology and to carry out large clinical studies, and (b) continuing education initiatives in teaching and research to exploit the growing assessment capabilities provided by technology.Technical barriers are due to (a) sEMG systems considered unfriendly, (b) the lack of familiarity with hardware and signal processing/interpretation techniques, and (c) the demand for fool-proof automatic equipment. The demand for support in the interpretation of signals and automatic warning of potential misuse and acquisition errors cannot be fully satisfied. Automatic expert system are no substitutes for human expertise and competence and may be misleading. No device should be used without knowledge of its performance, limitations and misuse, and without user's critical competence. Education and research should be institutionally planned and provided, like in other fields, at the academic level, by training new figures with a strongly inter- and multi-disciplinary approach. They will, in turn, train a new breed of clinical operators able to manage technology and interact with engineers and manufacturers.Finally, economical barriers, including cost/benefit analysis, should be seriously considered to identify the most economically rewarding sEMG-based applications, thus turning boundaries into project specifications. This requires fellowships for training researchers and funding for support of large clinical studies whose results will lead to reduction of the economic burden of institutions paying for treatment costs.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cerebrospinal fluid (CSF) is one of the most valuable sources of diagnostic biomarkers in neurological diseases. In the present study high sensitivity shotgun mass spectrometry was used to characterise the CSF lipidome of patients with MS, GBS and controls with non-demyelinating diseases. The quantification of 222 CSF lipid molecular species revealed characteristic changes in the absolute and relative lipid concentrations in MS and GBS compared to the controls. For the GBS group, the fourfold elevation in the total lipid content was a discriminatory and a newly identified feature of PNS demyelination. In contrast, in MS, the accumulation of the myelin-derived cerebrosides represented a specific feature of demyelination. As a common feature of demyelination, we identified upregulated levels of lipid metabolic intermediates. We found strong positive correlation between total protein content and lipid concentrations in both diseases. By exploring the CSF lipidome we demonstrate usefulness of broad-range shotgun lipidomic analysis as a fast and reliable method of biomarker discovery in patients with demyelinating neurological disorders that might be a valuable diagnostic complement to existing examinations.Multiple sclerosis (MS) and Guillain MS is one of the most common diseases of the CNS, the incidence rate is 30 cases per 100,000 people globally but in Europe it reaches 80 cases per 100,000. The symptoms show substantial degree of heterogeneity with autonomic, visual, motor and sensory problems being the most common2. Although its cause is unknown, it is assumed to occur as a result of complex genetic, epigenetic and environmental factors. The diagnosis is based on the integration of clinical, imaging and laboratory findings. The majority of patients experience a relapsing–remitting type of MS, however, over time half of these patients enter a progressive phase. Currently, there is no known cure for MS, but research is ongoing looking for more effective and tolerable treatments.Multiple sclerosis (MS) is a chronic inflammatory disease, where demyelination specifically affects the central nervous system (CNS)–Barré syndrome (GBS) is a postinfectious, acute immune-mediated polyradiculoneuropathy, where demyelination occurs in the peripheral nervous system (PNS)4. The incidence of GBS is rare, the rate is 1–2 cases per 100,000 people. GBS is thought to be an autoimmune disease, and the onset of symptoms is preceded in two-thirds of cases by a bacterial or viral gastroenteritis or respiratory infection5, Campylobacter jejuni, Epstein–Barr virus, cytomegalovirus and Zika virus being among the most common infective agents. Curiously, recent reports suggested the possible association between acute COVID-19 infection and GBS6, therefore the incidence of GBS during outbreak of COVID-19 infection can be increased. GBS is characterised by acute onset and a rapidly progressing ascending type of motor weakness, paraesthesia which reaches maximum severity within 4 weeks, where ca. 25–30% of patients require artificial ventilation7. The diagnosis is based on clinical history, nerve conduction studies and analysis of the cerebrospinal fluid (CSF). To alleviate its course, intravenous immunoglobulin therapy or plasmapheresis is used. With appropriate supportive care the prognosis of GBS is good and leads to full recovery in the majority.Guillain11. CSF is one of the most valuable and accessible sources of diagnostic biomarkers in neurological diseases. CSF is not simply an ultrafiltrate of plasma but fills and flushes the transcellular space of brain and spinal cord. Therefore, both the abnormal plasma infiltration and the appearance of myelin degradation products, as major consequences of demyelination, can be tracked by the analysis of CSF. It is known that the plasma and brain lipidomes display characteristic differences. For example, high levels of the storage lipids cholesteryl ester and triglyceride are plasma characteristics13, whereas sulfatides, gangliosides and the myelin-specific cerebrosides, also referred to as hexosylceramides, are typical brain lipids15. At lipid species level, the brain is rich in disaturated, monounsaturated and docosahexaenoic acid (22:6)-containing phospholipid species such as phosphatidylcholine PC(16:0/16:0), PC(16:0/18:1) and phosphatidylethanolamine PE(18:0/22:6), respectively16. In contrast, plasma lipid profile is known for the abundance of linoleic acid (18:2)-containing species such as PC(16:0/18:2) or arachidonic acid (20:4)-containing species like PC(16:0/20:4)13. Given these characteristic differences, CSF lipidomics might provide valuable information with regard to the origin as well as the underlying mechanisms of demyelination.A common pathological feature in MS and GBS is the leakage of the blood–brain/blood-CSF or blood-nerve barriers17, which is approximately 300-fold lower than that of plasma17. The development of high sensitivity mass spectrometry-based approaches18 enabled the examination of the CSF lipidome19. However, insofar reports focused mainly on dementia22, and comprehensive CSF lipidomic reports dealing with MS and GBS are few and incomplete.From bioanalytical point of view, CSF is a challenging biological fluid due to its low 15 µg/mL total lipid contentSubstantial symptom heterogeneity in MS and fast disease progression in GBS necessitate exploration of new rapid methods of biomarker discovery. In this study we applied a direct injection-based mass spectrometry method for the fast and comprehensive assessment of the CSF lipidome that allows for the correlation of lipid data with available clinical parameters and identification of differences in the CSF lipidomic patterns of demyelination in MS and GBS compared to non-demyelinating diseases.3. We detected strongly elevated albumin index in the GBS group with 73% of cases exceeding the normal maximum value of 9. In patients with MS, intrathecal IgG synthesis leads to the elevation of IgG to albumin ratio in CSF compared to serum23. The IgG index exceeded the normal limit of 0.7 in 63% of cases, and the IgG blot was positive in 96% of cases.Of the 118 neurological patients recruited from the Hungarian population in the city of Szeged, 77 proved eligible to participate in an exploratory retrospective CSF analysis. Table 24. To present lipidomic data we calculated both absolute quantities (lipid nmol/CSF mL) and relative levels (mol% of polar lipids)  and high goodness of fit (R2) with  and highSubsequently, we compared the molecular species patterns for the control, MS and GBS groups using Van der Waerden post-hoc tests for pairwise multiple comparisons  species in the MS group compared with the controls . The elevation included practically all measured lipid classes  to 57.5 (33.3–79.7) nmol/CSF mL, ses Fig.  but its ses Fig. . The incses Fig. . The higNext, we analysed the potential correlations between clinical and lipidomic features using the Spearman’s rank correlation analysis. In the absence of age-matched controls, we calculated partial correlation coefficients to control for the effect of age. Nevertheless, we note that age has negligible effect on lipid metabolite levels  with a fairly narrow EDSS range (1–4). However, we note that the EDSS value of 8.5, found in a patient with primary progressive MS, appeared to associate with markedly higher levels for several lipid species throughout the whole lipidome. For the GBS group, several relative lipid concentration values, including sphingo-, glycero and glycerophospholipid species, displayed moderate correlation with Hughes Functional Grading Scale (HFGS) disability scores measured at nadir and/or discharge. Noteworthy, correlations were positive for plasma-derived phosphatidylethanolamine, lysolipid, diglyceride and triglyceride components, whereas negative correlation was found for brain-specific sulfatide and GM3 ganglioside species  and an improving group . We detected no statistically significant differences between these categories with respect to the clinically diagnostic biomarker parameters . Interestingly, brain-derived sulfatide and PC species showed higher relative levels, whereas plasma-derived lysolipid, diglyceride and triglyceride species displayed lower values in patients in the clinically improving group Fig. . It sugg26, on the assessment of the sphingolipidome27 or the global lipidome with a fatty acid-targeted approach28 in patients with MS, or on the polar metabolome from patients with MS29 and GBS30.In the present study we report about a rapid and comprehensive lipid species analysis of the CSF lipidome, an insofar understudied area in CSF biomarker discovery. We presented the applicability of our method in this exploratory study that was aimed to better understand the role of lipidomic biomarkers in the pathology of demyelination in the CNS and PNS. There are only a few reports in the literature that analysed CSF lipids in MS or GBS. These studies were targeted either on SM analysis in demyelinating disorders of the PNS31 and GBS32. The consequences, i.e. the enhanced plasma infiltration and the augmented appearance of myelin-derived lipids in CSF are well distinguishable features by lipidomic analysis given the characteristic differences in the plasma and brain lipidomes. In general, we detected a distinctive change in the absolute and relative lipid amounts in MS and GBS as compared to controls with non-demyelinating disorders. This elevation suggests a characteristic alteration in lipid metabolism and/or lipid transport due to CNS or peripheral site of demyelination. The strong association between the total protein content and lipid levels represents a new piece of information for both diseases. For the GBS group, the massive elevation in lipid amounts was a distinctive and newly recognized feature of PNS demyelination. In addition to the highly elevated albumin index, the robust accumulation of plasma-derived lipids  as well as the imbalance in the composition of plasma-derived versus brain-derived lipid species confirmed the blood–brain/blood-CSF barrier damage and the abnormal leakage of plasma into the CSF. The elevation in SM agrees well with recently published results where targeted CSF lipid analysis revealed significantly higher SM content in patients with GBS compared to those with non-demyelinating disorders26. In the MS group, we could not observe increase in albumin index. This could be in agreement with reports that propose albumin index may not be an accurate measure of blood–brain barrier dysfunction in MS31. Nevertheless, at the level of the lipidome, the enrichment of plasma-specific triglyceride species might be an indication of enhanced plasma leakage into the CSF of patients with MS.It is a generally accepted view that the blood–brain/blood-CSF barrier is compromised in both MS27. In agreement with those findings, the palmitic acid-containing cerebroside species HexCer(34:1:2) was one of the best predictive markers for MS as compared to controls in our study too.On the other hand, the accumulation of sulfatides, ganglioside GM3 and cerebrosides in the GBS group, as well as the enrichment of cerebrosides in the MS group are indicative of demyelination. Considering the overall alterations in the CSF lipidomes, the selective accumulation of the myelin-specific cerebrosides seems to be a characteristic feature of CNS demyelination compared with that in the PNS. An increase in the levels of cerebrosides in the CSF of patients with MS has already been reported33. Other studies have demonstrated that Cer can be important mediator of oligodendrocyte death in the MS brain34. Furthermore, the activity of acid sphingomyelinase, which generates Cer from SM  appeared to be a promising candidate biomarker of disease progression in MSLimitations of the present study include the relatively small size of the study population and that recruitment of patients was from a geographically restricted area of Europe. Nevertheless, the study groups represented well the neurological disorders in focus with regard to basic demographic and clinical parameters. Therefore, our sample was justifiably suitable to perform an exploratory lipidomic study. An additional limitation is the absence of age-matched controls; nevertheless, the correlation analysis showed that age-dependence of lipid metabolite levels was negligible in our cohort. The predictive and especially the prognostic power of the CSF lipidomic analytical results should be verified in a larger cohort study in the future. Assessment of higher gangliosides, parallel plasma lipidomics and correlation of lipidomic data with clinical imaging results could further improve the understanding of the molecular mechanisms behind these demyelinating disorders.14, therefore, lipid analysis might be of valuable diagnostic value in demyelinating disorders. Here we provided evidence for the massive increase in CSF lipid content as well as for the relative accumulation of plasma-derived over brain-derived lipids as newly identified CSF lipidomic features in patients with GBS. These results revealed that abnormal plasma infiltration into CSF dominates the pathology of PNS demyelination. In contrast, in the MS group, the selective elevation in the amount of myelin-derived cerebrosides reports about a dominantly myelin degradation-driven pathology in the CNS. In addition, our data suggest the activation of different phospholipases, likely as a result of the inflammatory response at the site of the blood–brain/blood-CSF barrier damage. CSF total protein content showed strong positive associations not only with the total lipid content but also with the levels of several individual lipid species in both MS and GBS. This finding provides a good basis for further investigations in which CSF lipidomics together with the analysis of individual CSF proteins might reveal the role of specific lipid-protein interactions in the pathology of demyelination.The dry mass of both CNS and PNS myelin is characterized by a high proportion of lipids (70 to 85%)The low lipid content, accompanied by relatively high salt concentration, makes CSF a challenging biofluid from lipidomic point of view. Most studies apply chromatography-coupled methods for analytical purposes. Nevertheless, our data demonstrate usefulness of the direct injection-based shotgun strategy that might enable cross contamination-free, high throughput lipidomic analysis with broad-range coverage of the CSF lipidome. CSF shotgun lipidomics can therefore serve biomarker discovery in other neurological diseases beyond MS and GBS.The Regional Human Biomedical Research Ethics Committee has approved the analysis conducted in this study (135/2008). All examinations and experiments including lumbar puncture and CSF collection were carried out in accordance with the guidelines of the Declaration of Helsinki and were performed after obtaining written informed consent.Utilizing the CSF depository of the Department of Neurology , we performed retrospective analysis using 118 neurological patients recruited between 2007 and 2017 from the Hungarian population. CSF samples of patients with alcoholic diseases , acute infections , polyneuropathies , systemic autoimmune diseases , and cancer were excluded from the study. In addition, we diagnosed 9 patients with other demyelinating diseases . However, due to the low case number they could not be handled as a specific diseased control group and were also omitted from the study.48. The Expanded Disability Status Scale  was used to quantify disability at the time of sampling. Patients with GBS were classified according to the Brighton criteria49. The samples were collected within 1–49 days of symptom onset, before major therapeutic interventions . Motor functional deficits were retrospectively scored at nadir (lowest point of disease) and discharge (leaving for home or rehabilitation) by the Hughes Functional Grading Scale .Filtering left 77 participants eligible for this study including 22 patients with relapsing–remitting and 2 patients with primary progressive modality of MS, and 19 patients with the diagnosis of GBS . Disease modifying treatment was always started after the time of sampling in patients with MS or GBS. 34 patients with non-demyelinating disorders were considered as controls including patients with cerebral infarction (9), syncope (1), transient global amnesia (1), vertigo (2), headache (2), spinal injury (8), CADASIL (1), cerebellar ataxia (1), vascular myelopathy (2), cognitive impairment (3), glaucoma (1), amyotrophic lateral sclerosis (1), pseudodementia (1), and cerebral cavernoma (1). Patients with MS were in the acute relapsing stage of the disease and the diagnosis was confirmed using the McDonald’s diagnostic criteria50. Albumin index and IgG index were calculated as albuminCSF/albuminserum and CSF/serum, respectively.CSF and serum samples were obtained, processed, and analysed according to the international standardized biobanking consensus protocol of the BIOMS-Eu network51. The modifications included the formation of a one-phase solvent system (as in the Bligh and Dyer method52) before phase separation which ensures extensive lipid extraction. Moreover, we applied pure water instead of 0.2 M KCl upon phase separation induction. Because CSF contains enough salt to keep more polar lipids in the lower organic phase, this modification helped to reduce ion suppression, which is critical in shotgun lipidomics. To 250 μL of native CSF sample, 625 μL methanol containing 0.001% butylated hydroxytoluene (as antioxidant) and 0.5 μg di20:0-PC (as extraction standard) and 313 μL chloroform were added to form one phase . The mixture was left at room temperature for an hour with periodical vortexing, then 938 μL chloroform and 220 μL water were added for phase separation to occur ). After vortexing, the sample was centrifuged at 600 g for 10 min and the lower chloroform phase was evaporated. The residue was reconstituted in 300 μL chloroform:methanol:isopropanol  containing a mass spectrometry internal standard mix. The standard mix composition was adjusted to CSF lipid profile  and Optima LCMS grade . Lipid standards were obtained from Avanti Polar Lipids  and Nu-Chek-Prep . All other chemicals were purchased from Sigma-Aldrich  and were of the best available grade. Lipids from fresh-frozen CSF samples were extracted by using a modified version of the standard two-phase Folch extraction53. For mass spectrometry measurements, to 60 μL reconstituted lipid extract 4 μL dimethylformamide  or to 30 μL reconstituted lipid extract 34 μL 17.5 mM ammonium chloride  was added. 10 μL of these solutions was injected and data were acquired for 3 min. Acquisitions were performed at the mass resolution Rm/z 400 = 240,000. All CSF lipid extracts were measured in one batch (within 3 days), and the instrument was fully calibrated before batch processing. A pooled extract was prepared from the control samples and was used as a QC sample injected after every tenth sample54; all 8 QC injections fell within the 95% confidence interval after PCA analysis run on test  and injection QC samples . For sample delivery we applied a robotic nanoflow ion source TriVersa NanoMate  using chips with spraying nozzles having a diameter of 5.5 μm. The ion source was controlled by Chipsoft 8.3.1 software. The ionization voltages were + 1.3 kV and − 1.9 kV in positive and negative mode, respectively, and the backpressure was set at 1 psi in both modes. The temperature of the ion transfer capillary was 330 °C. These conditions, together with the applied infusion solvent composition and additive types, ensured degradation-free survey scans as validated previously55. Sum formulas for glycero(phospho)lipids are defined as the lipid class abbreviation followed by the total number of carbons and total number of double bonds for all chains, e.g. PC(34:1), whereas the lipid class(sn-1/sn-2) format specifies the structures and regiochemistry of fatty acyl side chains, e.g. PC(16:0/18:1). Sum formulas for sphingolipids are defined as the lipid class abbreviation followed by the total number of carbons, total number of double bonds and total number of hydroxyl groups in the long chain base and the fatty acyl moiety, e.g. SM(34:1:2). Sphingosine, SPH(d18:1), was considered as the major sphingoid base in which first the number of hydroxyl groups is designated by a letter (e.g. “d” for two hydroxyls) followed by the number of carbons and double bonds. In higher sphingolipids, the lipid class(sphingoid base/fatty acyl) format specifies the resolved structure, e.g. SM(36:1:2) corresponds to SM(d18:1/18:0).To annotate lipid classes and species, we applied the classification systems for lipidsLipidomic data are expressed either as absolute quantities (lipid nmol/CSF mL) or as relative levels. Relative levels were calculated as mol% of polar lipids, where polar lipids include glycerophospholipids and sphingolipids but not diglycerides and storage lipids. Absolute concentrations were calculated based on the amount of the extraction standard.56. Subsequently, Van der Waerden post-hoc tests were performed for pairwise multiple comparisons. We calculated the nonparametric effect size index A, which equals the area under a receiver operating characteristic curve (AUC) using the trapezoidal method and estimates the probability that a member of one population scores higher than a member of another population. A = 0.5 represents no change, whereas 0 and 1 represent maximal effects, decrease and increase, respectively57. A1,2 = 1 − A2,1 where the indexes of A refer to the two populations to be compared. A is not sensitive to group sizes and is much more robust to unequal variances or outliers58. False discovery rate  was determined according to the Storey Tibshirani method59. We calculated partial correlation coefficients to describe associations between clinical and lipid data with the control for age using Spearman’s rank correlation. These analyses were performed using R 3.6.3 60. Orthogonal partial least squares discriminant analysis (OPLS-DA) on lipidomic datasets was performed with MetaboAnalyst61.Demographic, clinical, and lipidomic data are presented as median and interquartile range. Because lipidomic data did not fulfil the assumptions of normality (Shapiro–Wilk test) and homoscedasticity (F test), we applied the Van der Waerden test for between-subjects analysis of varianceSupplementary Tables.Supplementary Figures.


In the rest position, excellent reliability was found for RMS and ApEn  and good to excellent reliability was found for MDF and MNF . The intra-session (test) and inter-session (re-test) analyses demonstrated the reliability of nonlinear sEMG variables of the masticatory muscles in adults with Down Syndrome.The aim of the present study was to evaluate intra-day (test) and inter-day (re-test) reliability of surface electromyography (sEMG) signals of the masseter and temporal muscles in patients with Down syndrome (DS). We determined the reliability of sEMG variables in 33 patients with DS. EMG signals were recorded at rest as well as during maximum voluntary clenching and maximum habitual intercuspation (MHI). The signals were analyzed considering the amplitude in the root mean square (RMS), mean frequency (MNF), median frequency (MDF) and approximate entropy (ApEn). The intraclass correlation (ICC This condition is associated with cognitive impairment, several comorbidities and emotional-social limitations, leading to substantial medical and social costs6.Down syndrome (DS), also known as trisomy of chromosome 21 (HSA21), is the most common genetic alteration, the prevalence of which ranges from six to 13 per every 10000 people in the general populatio7. The combination of abnormal masticatory muscle function (due to hypotonia) and altered skeletal developmental  has several consequences during the growth phase, such as oromotor incoordination , difficulties during meals , uncontrolled facial movements, mouth open at rest and mouth-breathing due to poor muscle tonicity10.Generalized muscular hypotonia is one of characteristics of DS, which, along with oral abnormalities, directly affects oral functions, such as swallowing, speech and breathing Some of these abnormal muscle functions, such as those in the muscles responsible for the sustaining and moving the jaw  can be measured using surface electromyography (EMGs). However, the selection of a measure for research or clinical use depends on several factors, such as reliability. In clinical practice, the combination of linear and nonlinear measures is important to the characterization, classification and evaluation of the complexity of sEMG variables of the masticatory muscles and can enable a better understanding of neurophysiological conditions.12. Median frequency (MDF) is another linear variable widely used to interpret spectral characteristics of sEMG signals, reflecting muscle electrophysiology in different clinical conditions such as temporomandibular disordes13, cerebral palsy14 and low back pain15.The root mean square (RMS) is a linear variable used to evaluate the excitability and activation of muscles before and after therapy16. Information entropy has been proposed as a measure of irregularity (nonlinear behavior) in biological signals19. The measurement of entropy is reported to be a reliable method for characterizing neuromuscular changes20 and approximate entropy (ApEn) enables a general understanding of the complexity of sEMG data22. In statistics, ApEn is used to establish the uncertainty or variability in a system. ApEn calculated from sEMG signals is dependent on the variability in both amplitude and frequency and better represents this aspect than amplitude or frequency alone22. As studies have demonstrated that nonlinear time-series analysis is more sensitive to changes in myoelectrical signals in comparison to linear methods24, the use of ApEn to quantify the irregularity or complexity of sEMG signals of the masseter and anterior temporal muscles in patients with DS may be important to the quantification and understanding of the neurophysiological conditions of these muscles.Nonlinear time-series analyses indicate features of motor control that are important for clinicians to measure25. This is a key factor to evaluating sEMG data in rehabilitation programs and clinical trials. Factors such as the preparation of the skin and positioning of the electrodes can exert an influence on the results when sEMG data are collected on different days27, as occurs when signals are collected before and after a clinical intervention.However, the usefulness of sEMG data is dependent on the reproducibility of the signal detection method both within and between recording sessions28, to the best of our knowledge, no studies have assessed the reliability of sEMG signals of the masticatory muscles or the reproducibility of sEMG variables (linear and nonlinear) of the masseter and anterior temporal muscles in adults with DS. It is important to determine the reproducibility of measuring sEMG signals (intra-session and inter-session reliability) prior to suggesting the use of this technique as a tool for evaluating the efficacy of therapy designed to improve masticatory muscle function in this population.Although a number of studies have demonstrated the reliability and reproducibility of sEMG signals for the evaluation of the masticatory muscles in both healthy and disabled patientsTherefore, the aim of the present study was to determine the intra-session (test) and inter-session (re-test) reliability of sEMG signals of the masseter and anterior temporal muscles in adults with DS.2 and mean neck circumference of 40.3 ± 4.2 cm (Table The sample was composed of 23 adults with DS (15 men and eight women) with a mean age of 22.7 ± 6.5 years, mean body mass index (BMI) of 28.5 ± 6.8 kg/mThe reliability of the sEMG variables was determined in all participants. Tables The present study confirmed the reliability  of different sEMG variables of the masseter and anterior temporal muscles recorded during MHI and in the rest position in adults with DS. The results of the linear (amplitude and frequency) and non-linear (ApEn) analyses revealed excellent intra-session and inter-session reliability of sEMG signal readings during MIH Table  as well 17. A high degree of reliability during MHI has also been found for the anterior temporal and masseter muscles in healthy patients30. These results demonstrate that sEMG activity recorded during MIH and at rest can be used to quantify the effects of clinical interventions on the masseter and anterior temporal muscles.These results are similar to those reported in a previous study involving patients with cerebral palsy, i.e., high reproducibility for the data recorded during MIH and good to excellent reproducibility for data recorded in the rest position32 or for the observation of muscle activity in the clinical setting30. Multiparameter sEMG analysis has been used as a strategy to investigate the performance of the masticatory muscles following a stroke34, assess pain patterns in the masticatory muscles35 and investigate the reproducibility of the evaluation of the masticatory muscles in patients with cerebral palsy17.The sEMG evaluation is diagnostically useful in identifying patients with pain-related temporomandibular disorders22. Specifically for ApEn, the result is given between 0 and 2, with higher values reflecting greater irregularity within the time-series38. Thus, values near 2 indicate healthy muscle function. The excellent reliability found for ApEn under both test conditions (MIH and rest) could be an important stimulus for future studies to use this sEMG parameter in the evaluation of the effects of therapeutic interventions performed on the masseter and anterior temporal muscles.Electromyographic components calculated in the time and frequency domains are the most widely used variables for the study of masseter and anterior temporal muscle activity, whereas nonlinear components, such as ApEn, remain under-investigated in clinical studies. This is an issue that needs to be better explored, as a change in the entropy of the signal also reflects a qualitative change in the physiological systemThe present findings demonstrate the excellent reliability of surface electromyography variables  of the masseter and anterior temporal muscles in adults with Down Syndrome during maximum clenching effort as well as good to excellent reliability of the median and mean frequency recorded in the rest position.A convenience sample of adult patients with Down syndrome were evaluated at the Center of Bioscience Applied to Persons with Special Care Needs (CEBAPE), Institute of Science and Technology – UNESP/SJC, Brazil. The inclusion criteria were adult patients aged between 18–35 years-old, presenting snoring and/or mild to moderate apnea/hypopnea index, preserved cognitive function , agreed to participate by free will and, the Awareness Term along with signed by patient or patient’s responsible. The exclusion criteria were body mass index (BMI) greater than 30, unsatisfactory dental health, not able to reach the University when is needed, present psychiatric disease and have been submitted to physiotherapy, speech therapy and/or orthodontic treatment at least 6 months before the beginning of this study.This study received approval from the Human Research Ethics Committees of the Institute of Science and Technology– UNESP/SJC, Brazil, CEPh 2.127.141 (process number CAAE 64173616.4.0000.0077).The device used for the EMG acquisition was an eight-channel module (EMG System do Brasil Ltda®) consisting of a conditioner with a band pass filter with cut-off frequencies at 20 to 500 Hz, an amplifier gain of 1000 times and a common mode rejection ratio >120 dB. All data were acquired and processed using a 16-bit analog to digital converter (EMG System do Brasil Ltda®) with a sampling frequency 2 kHz. Active bipolar electrodes with a pre-amplification gain of 20x are included.The participants visited the Physiology Laboratory on two different occasions, with a one-week interval between visits. Sessions were held in a silent, well-lit recording room. The participant was instructed to remain seated comfortably erect on a chair with eyes open, feet apart, shoulders relaxed and hands resting on thighs, with the head on the Frankfort plane parallel to the ground. During the procedures, the participants did not receive any visual feedback of the signals displayed on the computer.A short training period was conducted prior to the tests to familiarize the participant with the tasks. Explanations concerning the procedures and electrode placement were given and the participant was trained to bite as hard as possible with maximum voluntary clenching (MVC) effort with a cotton role between the arches and maximum habitual intercuspation (MHI) effort with no material interposed between the teeth.13. Bandage tape was used to secure the electrodes further, with care taken to avoid micro movements. A rectangular metallic electrode measuring 3 ×2 cm coated with Lectron II conductive gel  to increase the conduction capacity and impede interference from external noise was attached to the left wrist as a ground reference.The sites for the placement of the electrodes were shaved and cleaned with a cotton ball soaked in 70% alcohol to diminish impedance between the skin and electrode. Pre-gelled, self-adhesive, bipolar, disposable, Ag/AgCl surface electrodes (MediTrace®) measuring 10 mm in diameter were positioned over the right masseter (RM), left masseter (LM), right temporal (RT) and left temporal (LT) muscles with an inter-electrode distance of 20 mm in the region of greatest tonus after the participant performed moderate intercuspationReadings were performed under three conditions: at rest, during MVC and during MHI. In session 1 (test), three readings (10 seconds each) were performed in the rest position, followed by three readings (five seconds each) during MVC with a two-minute interval between readings. After three minutes, three readings (five seconds each) were performed during MIH with a two-minute interval between readings. The same procedures were repeated after one week in session 2 (retest).17. For the analysis of MVC and MIH, the first and last seconds of the five-second signals were discarded, resulting in three-second readings considered for each test. In contrast, all 10 seconds of the signals captured in the rest position were considered  and frequency domains as well as nonlinear analysis through the calculation of the complexity of the sEMG signalThe amplitude of sEMG signal was determined considering root mean square (RMS) using a 200-ms moving window. The mean RMS values of the three readings at rest and during MIH were normalized by the highest RMS values obtained during the three MVC readings (µV/µV × 100: % MVC).17 and the average of the values among the three windows was used in the reliability tests.The power spectral density of the sEMG signals was calculated using Welch’s averaged periodogram with a Hamming window length zero-padded to the length of 2048 points. Overlap was 50% of the window length. The mean frequency (MNF) and median frequency (MDF) of the power spectrum were calculated for each one-second window 38. This analysis returns a value between 0 and 2 (arbitrary units [au]), with higher values reflecting greater irregularity in the time series38.ApEn  was calculated to quantify the regularity or complexity of the sEMG signals, with the embedding dimension (m) and tolerance distance (r) set to m = 2 and r = 0.20 of the standard deviation (± SD) of the data sequence and N = number of points to be analyzedThe sEMG signals were processed using specific routines carried out in Matlab version 7.1 .2,1) using only the first and second of the three measurements. Inter-day reliability  was determined by comparing the average (3 trails) of each sEMG activity index for each muscle between the two sessions (test and retest). SEM was estimated by subtracting the ICCs value from one, taking the square root of this value, and multiplying by the SD39. The ICCs was interpreted using the following criteria: 0.00 to 0.39 (poor), 0.40 to 0.59 (fair), 0.60 to 0.74 (good) and 0.75 to 1.00 excellent)40. The SEM was used to express reliability in absolute values, with a lower SEM denoting greater reliability of the measurement, whereas a high SEM indicates a high level of error and implies the non-reproducibility of the tested values.The Shapiro-Wilk test was used to determine the distribution of the data and data were expressed as mean and SD. Intraclass correlation coefficients (ICCs) and the standard error of the mean (SEM) was used to verify the reliability of each sEMG measure. Intra-session reliability was determined by computing the intraclass correlation coefficient  Version 17.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee  and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed written consent, for adult with Down syndrome in this study, was obtained from their parent/caregiver.


Intracranial hypotension (IH) is a relatively common condition associated with low cerebrospinal (CSF) pressure. The most typical symptom is orthostatic headache, although neurological deficits and changes in the level of consciousness, such as encephalopathy, stupor, and coma, may also occur. Uncomplicated CSF hypotension headaches generally resolve with rest, hydration, and analgesia. However, persistent cases may require an epidural blood patch (EBP) for resolution. Our report presents the case of a 50-year-old male with a history of intravenous (IV) drug abuse, positive for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) antibodies, who was admitted for new-onset headache and brain magnetic resonance imaging (MRI) findings suggesting CSF hypotension. The patient subsequently developed altered mental status with agonizing respirations, prompting intubation and admission to the intensive care unit (ICU) with neurosurgery consult. The initial exam revealed fixed and dilated pupils, suggestive of severe IH with brain herniation and the decision was made to proceed with an emergent intrathecal infusion with intraparenchymal intracranial pressure (ICP) monitoring, combined with EBP. A substantial clinical improvement was noted following the procedure. Within 45 minutes, the patient’s mental status improved to normal and pupillary dilation and areflexia were no longer observed. While the procedure may need to be repeated in cases of late deterioration, this report provides evidence that intrathecal bolus saline infusion with simultaneous ICP monitoring may be considered an effective measure to treat extreme cases of IH with associated brain herniation. If performed in a timely fashion, improvement of ICP numbers, and clinical resolution can be quite rapid. Intracranial hypotension (IH) is commonly caused by a traumatic cerebrospinal fluid (CSF) leak secondary to lumbar puncture or surgery but may also arise spontaneously. IH is a relatively common condition associated with low CSF pressure, with the most common presentation of orthostatic headache. However, other symptoms and even neurological deficits may be present, including nausea, vomiting, diplopia, photophobia, hearing changes, ataxia, limb paresthesias, loss of bowel and bladder control, and changes in personality. Changes in the level of consciousness, including encephalopathy, stupor, and coma, may also occur -2. The constellation of symptoms likely depends upon the degree of hypotension. As the CSF pressure decreases, the naturally buoyant force that suspends the brain is decreased, causing the brain to sag . This maHerein, we present a particularly severe, life-threatening case of IH associated with headache and progressive neurological deterioration requiring intubation and mechanical ventilation. In order to achieve resolution, multiple epidural blood patches combined with intrathecal saline infusions were necessary.3 with evidence of lymphocytic pleocytosis, suggesting the possibility of aseptic meningitis for which he was placed on Acyclovir.A 50-year-old male was originally admitted to an affiliate hospital after his wife witnessed him having convulsions at home. He had a history of intravenous (IV) drug abuse and was positive for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) antibodies. A lumbar puncture was performed, and he was found to have a CSF white blood cell count of 65 cells/mmTwo days later, the patient developed a headache of 6/10 severity, not associated with nausea, vomiting, or visual changes. A brain MRI without contrast was performed, demonstrating new, bilateral holohemispheric subdural effusions with effacement of the sulci and distortion and downward displacement of the midbrain, most suggestive of interim development of CSF hypotension  without contrast was performed. In comparison to the previous study, it showed worsening diffuse subdural hemorrhage, largest along the left frontal convexity with a 1 - 2 mm right midline shift and low-lying cerebellar tonsils . Multiple EBP administrations may be necessary as well before an effect takes hold.


Embryo transfer has been successfully used for the conservation of equine endangered species, but a number of factors may affect the outcome of these techniques in mares. However, only a few studies have evaluated these factors in donkeys. The present study was conducted to determine which factors affect the recovery rate, morphological quality, and diameter in embryos from Andalusian donkey jennies. According to our results, the factors affecting embryo recovery rate were donor jenny, donor age, successive cycle within donor, number of flushings, and jack. Day of flushing and number of flushings had an effect on embryo diameter, whereas donor jenny and day of flushing had an effect on embryo quality. The knowledge of these factors is crucial to achieve a higher efficiency of embryo transfer in endangered donkey breeds.p = 0.040) and embryo recovery  as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR;  Equus africanus asinus) were used as pack animals in agricultural activities, commerce, and militia [In the past, domestic donkeys  [The conservation of endangered species is an excellent opportunity for applying assisted reproductive technologies such as embryo transfer, embryo cryopreservation, and germplasm cryobanking. Embryo transfer (ET) has been successfully used for the conservation of equine endangered species such as Przewalski´s horses  , and numwalskii) . Togethewalskii) ,14. Howewalskii) ,19,20,21It is known that some factors may affect the embryo recovery rate and embryo diameter and morphological quality in mares, including the day of flushing, number of ovulations, age of the donor, and quality of semen , and thaThe aim of the present study was to determine which factors affect the recovery rate, morphological quality, and diameter in embryos from Andalusian donkey jennies as a prerequisite to improve the success of both embryo transfer and cryopreservation in this endangered donkey breed.All animal procedures were approved by the Ethical Committee for Animal Experimentation of the University of Cordoba (no. 31/08/2017/105) and are in accordance with Spanish laws for animal welfare and experimentation .n = 5), 4–9 (n = 17), and ≥10 years old (n = 5). From February to December of three consecutive years (2015–2017), a total of twenty-six healthy Andalusian jennies (3–13 years old), of known fertility, served as embryo donors, and eight Andalusian jacks (6–9 years old) known to be fertile were used to mate the donors. To assess the effect of the age, the donor jennies were divided into three categories: ≤3  in the presence of corpus luteum. Donor jennys received human chorionic gonadotropin  to induce ovulation when a follicle of 35–40 mm was detected. Next day, donor jennies were bred by live cover every other day until ovulation.Ovarian activity was evaluated by transrectal ultrasonography  on a biweekly schedule during diestrus and daily during oestrus until ovulation (Day 0 = day of ovulation). Estrus was induced with one intramuscular injection of 5.25 mg luprostiol , as previously described [Six to nine days after ovulation, donor jennies were flushed 3 times with a total of 3 L of Lactated Ringer´s solution , as described by Camillo et al.  for donkescribed . The diaescribed .Descriptive statistical analysis was performed, presenting the qualitative variables as frequencies and percentages, and quantitative as a mean ± standard error of the mean (SEM). The effects of the year in which the study was performed (1–3), season , photoperiod , herd (1–3), days of flushing for embryo recovery (6–9), number of flushings (1–3), donor (25 jennies), donor age , parity (nulliparous vs. multiparous), successive cycle within donor , number of ovulations per cycle (single vs. double), and jack (8 donkeys) on positive uterine flushing rates , embryo recovery rate , and on ovulation rate  were analyzed by the Chi-square test and by the Kruskal–Wallis one-way ANOVA, respectively. When the effect was statistically significant, post-hoc multiple comparisons were made using Chi-square tests for categorical variables and Mann–Whitney U tests for continuous variables. To evaluate the effects of single factors  on embryo quality (Grade 1–4), diameter (µm), and developmental stage  the Kruskal–Wallis one-way ANOVA was performed. Mean values were compared by Duncan’s test.p < 0.05.All analyses were performed using the statistical package SPSS v15.0 . Differences were considered statistically significant when p = 0.01). Results of statistical analysis also showed differences among herds (p = 0.031), while no differences (p > 0.05) were observed among donor age categories, years, photoperiods, and seasons (p > 0.05) on both ovaries . However, the incidence of bilateral double ovulation (18.3%) was significantly higher (p < 0.01) than that of ipsilateral double ovulation (6.7%).The average ovulation rate in donor jennies is shown in  seasons . The sinA total of 124 uterine flushings were carried out during the study, of which 92 were positive , and 100 embryos were recovered out of 124 estrous cycles  and 157 ovulations .p < 0.05) varied according to developmental stage and day of recovery, being lower for blastocysts or when flushed at day 8 after ovulation (p < 0.001) by the developmental stage and day of flushing. The mean diameter of embryos was 179.39 ± 9.61 µm (range: 150–300 µm) for morulae, 210.81 ± 7.90 µm (range: 150–325 µm) for early blastocysts, 425.00 ± 45.32 µm (range: 275–600 µm) for blastocysts, and 1022.92 ± 125.22 µm (range: 250–3300 µm) for expanded blastocysts. Moreover, it was observed that embryos recovered at 6 days after ovulation had a diameter of 187.50 ± 15.23 µm (range: 150–300 µm); those collected on day 7 measured 236.48 ± 13.83 µm (range: 150–600 µm); while the mean diameter at 8 and 9 were 806.25 ± 83.31 µm (range: 275–2400 µm) and 2275 ± 300.14 µm (range: 1525–3300 µm), respectively  PFR or ERR; however, there was an effect of the number of flushings  on both rates, which were significantly (p < 0.001) reduced in the third flushing .p > 0.05) with any of the studied variables except the donor (p = 0.040). No differences between parity (p = 0.2610) and number of ovulations (p = 0.0971) were detected for ERR (p < 0.05) but also among donor age groups (p < 0.05) and successive cycles within the donor (p < 0.001). ERR was higher (p < 0.05) in jennies of 4–9 years of age  with respect to the other groups (≤3 years: 0.77 (26/34); ≥10 years: 0.64 (23/36)). With regard to the number of ovulations in the same donor, ERR was significantly higher (p < 0.001) in the first group  with respect to the second  and third  groups.The intrinsic factors that affect PFR and ERR are shown in  for ERR . In contp > 0.05) among jacks. Four jacks  that were used in 79.7% of cycles (94/118) showed good results for both rates . The other three jacks  that were used in 18.6% of cycles (22/118) yield a lower PFR (50–58.3%) and ERR (0.50–0.67) than the previous group, but this was not significant statistically (p > 0.05). No embryos were obtained with jack number 232, although he was used only two times. As shown in p < 0.05) these three variables, except for the day of flushing (6–9), which significantly influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos  and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different among herds  of the intrinsic factors on developmental stage, embryo quality, and diameter of embryos, except for the donor, which affected the embryo quality (p = 0.004). Moreover, no differences (p > 0.05) were detected among jacks for these three variables  routinely used in horses have been applied directly to donkeys. Hence, previous studies have demonstrated the suitability of mare ET techniques for collecting embryos in jennies . In thisIn this study, in which 26 donor jennies and 124 cycles were used, the average ovulation rate per jenny was 1.26 ± 0.04. This finding was slightly lower than the reported average in spontaneous (1.57 ± 0.06) and prostaglandin F2 alpha (PGF2α)-induced (1.56 ± 0.10) estrus of Andalusian jennies . These sIn jennies, the incidence of multiple ovulation was reported to range between 5.3% and 61% ,36,37. INo influence of the age of the donor on the ovulation rate was observed in this study; however, the ovulation rate was numerically (1.36 ± 0.10%), but not significantly higher, in the older jennies (≥10 years old). These results are consistent with previous findings in jennies ,30 and mp = 0.095). Similar findings have been reported in previous studies [p = 0.031), which could be explained by the individuals comprising each herd. In fact, this study pointed out the existence of significant differences among jennies (p = 0.01). The ovulation rate did not vary among years, seasons, and photoperiods, but it tended to be lower in summer  affected significantly PFR or ERR. Consistent with previous findings , the ERRp = 0.08), flushing the uterus 9 days after ovulation. Taken all together, our results emphasize the importance of collecting Day 6 to 7 donkey embryos, smaller than 300 µm in diameter and with good morphological score, to ensure vitrification success, as has been previously suggested [In donkeys, the influence of the day of flushing on ERR has not been well established in the literature ,20. Undeuggested . From a practical point of view, another interesting observation was the effect of the number of flushings  on both rates (PFR and ERR), which were significantly reduced in the third flushing , indicating that the majority of embryo collections require one or two maximum flushings. In addition, embryo diameter and developmental stage varied among flushings, with larger and older embryos in the first flushing. Thus, our results could suggest that the larger diameter of the older embryos could facilitate their recovery .The embryos recovered in herd 1 were in earlier developmental stages than in the other herds. These results were probably due to the fact that all the embryo rerecovered in this herd (5 embryos) were flushed 6–7 days after ovulation, while in herds 2 (56 embryos) and 3 (39 embryos), flushes were carried out from Day 6 to 9 after ovulation, thus increasing the average diameter and developmental stage of the recovered embryos. Regarding the intrinsic factors, PFR did not vary with any of the studied variables  except for the donor jenny. In contrast, ERR not only varied among donors but also among donor age categories, showing higher values in jennies of 4–9 years of age (0.94) compared with all the other categories of age. Moreover, both PFR and ERR were not different between younger (≤3 years old) and older (≥10 years old) jennies. Our findings are in contrast with the results of previous studies ,20, whicThe effect of repeated uterine flushings has been previously described in mares , which wp = 0.0971). It has been also reported that ipsilateral double ovulations resulted in a lower ERR than bilateral double ovulations [p = 0.196).It has been previously described that the occurrence of multiple ovulations enhances ERR in mares ,51 and dulations , which culations ,53. In tConversely, the number of ovulations in the same donor influenced ERR, being significantly higher in the first group , but no effect on PFR was observed. These results partially agree with the findings of Camillo et al. , who obsBased on our results, we can conclude that the donor jenny was the main factor that affects the rate of positive flushings and recovery rates as well as the embryo quality. Other factors that affected embryo recovery rate were the number of flushings, donor age, successive cycle within donor, and jack. Meanwhile, the rate of positive flushings was affected by the number of flushings and the jack. From a practical point of view, these findings could indicate that the majority of embryo collections require one or two maximum flushings per cycle. Moreover, the negative effect of repeated uterine flushings on embryo recovery rate was proven, being lower after six consecutive cycles. On the other hand, the day of flushing had a significant effect on embryo quality and diameter, which emphasizes the importance of collecting Day 6 to 7 donkey embryos, with good morphological score and smaller than 300 µm in diameter, if embryos are going to be cryopreserved.