Nathalie Lévêque
email: nathalie.leveque@aphp.fr

Anneke Slis

Leonardo Lancia

Gaëlle Bruneteau

Cécile Fougeron

Acoustic Change Over Time in Spastic and/or Flaccid Dysarthria in Motor Neuron Diseases

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Motor neuron diseases (MND) embrace a group of neurodegenerative diseases affecting lower motor neuron (LMN) and/or upper motor neuron (UMN) systems. One of the diseases that involves the LMN system is Spinal and Bulbar Muscular Atrophy (SBMA), also known as Kennedy's disease [START_REF] Kennedy | Progressive proximal spinal and bulbar muscular atrophy of late onset. A sex-linked recessive trait[END_REF]. Primary Lateral Sclerosis (PLS) purely affects the UMN system [START_REF] Gordon | The natural history of primary lateral sclerosis[END_REF] and Amyotrophic Lateral Sclerosis (ALS) is defined as a disease affecting both systems resulting in heterogenous clinical profiles depending on the predominance of LMN or UMN involvement [START_REF] Charcot | Deux cas d'atrophie musculaire progressive : Avec lésions de la substance grise et des faisceaux antéro-latéraux de la moelle épinière[END_REF][START_REF] Ince | Amyotrophic lateral sclerosis: Current issues in classification, pathogenesis and molecular pathology[END_REF]. The major challenge in clinical practice is the lack of biomarkers to validate LMN and UMN involvement [START_REF] Bede | Editorial: Biomarkers and Clinical Indicators in Motor Neuron Disease[END_REF]. SBMA and PLS are frequently misdiagnosed as ALS, at least at onset of the disease. A correct distinction between these diseases is of utmost interest because the median disease duration until death is much longer in PLS [START_REF] Pringle | Primary lateral sclerosis. Clinical features, neuropathology and diagnostic criteria[END_REF] and in SBMA [START_REF] Atsuta | Natural history of spinal and bulbar muscular atrophy (SBMA): A study of 223 Japanese patients[END_REF] than in ALS [START_REF] Logroscino | Descriptive epidemiology of amyotrophic lateral sclerosis: New evidence and unsolved issues[END_REF]. It is crucial to find objective and reliable tools to assist the neurologic differential diagnosis, to monitor and predict disease progression across MNDs.

When the lower and/or upper motor neurons, which both innervate the orofacial and laryngeal regions, are damaged, motor speech function is altered. As a result, dysarthria is common in these three pathologies [START_REF] Atsuta | Natural history of spinal and bulbar muscular atrophy (SBMA): A study of 223 Japanese patients[END_REF][START_REF] Banno | Swallowing markers in spinal and bulbar muscular atrophy[END_REF][START_REF] Bede | Revisiting the pathoanatomy of pseudobulbar affect: Mechanisms beyond corticobulbar dysfunction[END_REF][START_REF] Finegan | The clinical and radiological profile of primary lateral sclerosis: A population-based study[END_REF][START_REF] Miyata | The facial nerve atrophy with spinal and bulbar muscular atrophy patients (SBMA): Three case reports with 3D fast imaging employing steady-state acquisition (FIESTA)[END_REF][START_REF] Pedroso | Perioral and tongue fasciculations in Kennedy's disease[END_REF][START_REF] Praline | Pure bulbar motor neuron involvement linked to an abnormal CAG repeat expansion in the androgen receptor gene[END_REF][START_REF] Querin | Biomarkers of Spinal and Bulbar Muscle Atrophy (SBMA): A Comprehensive Review[END_REF][START_REF] Tomik | A phenotypic-genetic study of a group of Polish patients with spinal and bulbar muscular atrophy[END_REF][START_REF] Turner | Primary lateral sclerosis: Consensus diagnostic criteria[END_REF][START_REF] Yunusova | Clinical Measures of Bulbar Dysfunction in ALS[END_REF]. The Mayo Clinic system (Darley et al., 1969a(Darley et al., , 1969b[START_REF] Darley | Multiple Orofacial Indices in Amyotrophic Lateral Sclerosis[END_REF] classifies dysarthria perceptually into several subgroups, based on the underlying neuropathology. Speech impairment, associated with LMN lesions, such as in SBMA, is categorized as flaccid dysarthria; speech impairment due to UMN damage, such as in PLS, is typically classified as spastic dysarthria. And finally, ALS is classified as mixed flaccid-spastic dysarthria, involving both LMN and UMN impairment.

The degree of spasticity and flaccidity affecting the orofacial and laryngeal regions depends in this latter case on the extent of damage of the UMN and LMN systems respectively [START_REF] Tomik | Dysarthria in amyotrophic lateral sclerosis: A review[END_REF]. In the Mayo Clinic classification, articulatory distortions, described as distorted vowels and imprecise consonant productions, figure among the most deviant speech characteristics encountered in these three dysarthria subtypes. Slow rate is also described to be a prominent feature in both spastic and flaccid-spastic dysarthria subtypes.

In the field of motor speech disorders, there are ongoing efforts to identify acoustic biomarkers to support the differential diagnosis in dysarthria. The motivation of this study is that, to our knowledge, no study compared the nature of dysarthric speech deficits in these three neurodegenerative MNDs. Hence, the current study investigates acoustic parameters as biomarkers to differentiate dysarthria subtypes between these three MNDs.

Acoustic and kinematic studies examining flaccid dysarthria subtypes due to different etiologies have shown several important articulatory, spectral, and temporal characteristics.

Flaccid dysarthria is explained by muscular weakness involving the lingual, laryngeal and velopharyngeal systems. In this broad subtype of flaccid dysarthrias originating from different lesions in the motor unit, speech impairments are usually characterized by velopharyngeal insufficiency, resulting in hypernasality, distorted labial and/or lingual phonemes and distorted vowels. Slow speech rate and slow labial and/or lingual alternating motion rates have also been described. Yet, reduced speech rate is usually not perceived in flaccid dysarthria, unless impairment is severe [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF]. In SBMA specifically, hypernasality has been shown to be significantly higher in patients compared to controls and increased vocal cords adduction during phonation has been observed as well, which is associated with laryngospasms [START_REF] Tanaka | Distinct acoustic features in spinal and bulbar muscular atrophy patients with laryngospasm[END_REF][START_REF] Tanaka | Nasometric Scores in spinal and bulbar muscular atrophy-Effects of palatal lift prosthesis on dysarthria and dysphagia[END_REF]. Although muscular atrophy and weakness of the tongue have been related to swallowing troubles in SBMA [START_REF] Banno | Swallowing markers in spinal and bulbar muscular atrophy[END_REF][START_REF] Hashizume | Quantitative Assessment of Swallowing Dysfunction in Patients with Spinal and Bulbar Muscular Atrophy[END_REF][START_REF] Mano | Tongue pressure as a novel biomarker of spinal and bulbar muscular atrophy[END_REF][START_REF] Mano | Head Lift Exercise Improves Swallowing Dysfunction in Spinal and Bulbar Muscular Atrophy[END_REF], to our knowledge, acoustic and articulatory studies investigating the effect of SBMA on the articulatory dimension do not exist.

Acoustic and articulatory studies examining spastic dysarthria due to various etiologies, demonstrate slow and incomplete velopharyngeal and lingual movements, reduced speech rate, increased duration of phoneme-to-phoneme transitions, reduced alternating motion rates, reduced speed and range of tongue and jaw movements, reduced rate and slope of F2 transitions, smaller vowel space and centralization of vowel formants [START_REF] Charcot | Deux cas d'atrophie musculaire progressive : Avec lésions de la substance grise et des faisceaux antéro-latéraux de la moelle épinière[END_REF][START_REF] Hirose | Patterns of Dysarthric Movement in Patients with Amyotrophic Lateral Sclerosis and Pseudobulbar Palsy[END_REF][START_REF] Kent | Acoustic studies of dysarthric speech: Methods, progress, and potential[END_REF][START_REF] Ozawa | Symptomatic differences in decreased alternating motion rates between individuals with spastic and with ataxic dysarthria: An acoustic analysis[END_REF][START_REF] Portnoy | Diadochokinetic syllable rate and regularity in normal and in spastic and ataxic dysarthric subjects[END_REF][START_REF] Rong | Relationship between kinematics, F2 slope and speech intelligibility in dysarthria due to cerebral palsy[END_REF][START_REF] Roy | A Description of Phonetic, Acoustic, and Physiological Changes Associated With Improved Intelligibility in a Speaker With Spastic Dysarthria[END_REF][START_REF] Schölderle | Dysarthria in Adults With Cerebral Palsy: Clinical Presentation and Impacts on Communication[END_REF][START_REF] Ziegler | Spastic dysarthria after acquired brain injury: An acoustic study[END_REF]. Many of these observations, however, were based on studies including one or a few speakers [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF], and articulatory impairments specifically arising in speakers with PLS have not been investigated yet.

Articulatory deficits in ALS have been examined the most extensive. Dysarthria associated with ALS can be either flaccid or spastic at onset, becoming mixed spastic-flaccid as dysarthria progresses. Both acoustic and kinematic studies report impaired articulatory range of movement with flattened F2 slopes and reduced vowel space, decreased range of movement of the tongue and exaggerated range or the movement of the jaw [START_REF] Darley | Multiple Orofacial Indices in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Kuruvilla-Dugdale | An investigation of clear speech effects on articulatory kinematics in talkers with ALS[END_REF][START_REF] Susan | Physiologic Deficits in the Orofacial System Underlying Dysarthria in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Mefferd | A novel fixed-target task to determine articulatory speed constraints in persons with amyotrophic lateral sclerosis[END_REF][START_REF] Rong | Predicting Early Bulbar Decline in Amyotrophic Lateral Sclerosis: A Speech Subsystem Approach[END_REF][START_REF] Shellikeri | Speech Movement Measures as Markers of Bulbar Disease in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Articulatory Movements During Vowels in Speakers With Dysarthria and Healthy Controls[END_REF], 2010, Lee et al., 2018[START_REF] Lenth | emmeans: Estimated Marginal Means, aka Least-Squares Means[END_REF]Lee & Bell, 2018;[START_REF] Mefferd | Tongue-and Jaw-Specific Articulatory Changes and Their Acoustic Consequences in Talkers With Dysarthria due to Amyotrophic Lateral Sclerosis: Effects of Loud, Clear, and Slow Speech[END_REF][START_REF] Rong | Automatic extraction of abnormal lip movement features from the alternating motion rate task in amyotrophic lateral sclerosis[END_REF][START_REF] Shellikeri | Speech Movement Measures as Markers of Bulbar Disease in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Kinematics of disease progression in bulbar ALS[END_REF][START_REF] Yunusova | Tongue Movements and Their Acoustic Consequences in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Speech in ALS: Longitudinal Changes in Lips and Jaw Movements and Vowel Acoustics[END_REF]. The range of movement of the tongue was shown to decrease across the severity continuum of dysarthria (Lee et al., 2018[START_REF] Lee | Direction-Specific Jaw Dysfunction and Its Impact on Tongue Movement in Individuals With Dysarthria Secondary to Amyotrophic Lateral Sclerosis[END_REF]Lee & Bell, 2018;[START_REF] Mefferd | Tongue-and Jaw-Specific Articulatory Changes and Their Acoustic Consequences in Talkers With Dysarthria due to Amyotrophic Lateral Sclerosis: Effects of Loud, Clear, and Slow Speech[END_REF][START_REF] Rong | Automatic extraction of abnormal lip movement features from the alternating motion rate task in amyotrophic lateral sclerosis[END_REF][START_REF] Shellikeri | Speech Movement Measures as Markers of Bulbar Disease in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Kinematics of disease progression in bulbar ALS[END_REF][START_REF] Yunusova | Tongue Movements and Their Acoustic Consequences in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Speech in ALS: Longitudinal Changes in Lips and Jaw Movements and Vowel Acoustics[END_REF].

Whereas [START_REF] Slis | Analysing spectral changes over time to identify articulatory impairments in dysarthria[END_REF] found higher variability in acoustic energy change, indicating more variability in vocal tract shape change, variability of the tongue, lips and jaw movements were demonstrated to be reduced in several other studies [START_REF] Kuruvilla-Dugdale | An investigation of clear speech effects on articulatory kinematics in talkers with ALS[END_REF][START_REF] Kuruvilla-Dugdale | Spatiotemporal movement variability in ALS: Speaking rate effects on tongue, lower lip, and jaw motor control[END_REF][START_REF] Mefferd | Speaking rate effects on articulatory pattern consistency in talkers with mild ALS[END_REF]. Acoustic and kinematic studies also showed impaired temporal aspects of speech rate and articulatory movements [START_REF] Mefferd | Articulatory-to-Acoustic Relations in Talkers With Dysarthria: A First Analysis[END_REF][START_REF] Mefferd | Tongue-and Jaw-Specific Articulatory Changes and Their Acoustic Consequences in Talkers With Dysarthria due to Amyotrophic Lateral Sclerosis: Effects of Loud, Clear, and Slow Speech[END_REF][START_REF] Shellikeri | Speech Movement Measures as Markers of Bulbar Disease in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Yunusova | Articulatory Movements During Vowels in Speakers With Dysarthria and Healthy Controls[END_REF], 2010, 2012, Ball et al., 2002;[START_REF] Mefferd | Speaking rate effects on articulatory pattern consistency in talkers with mild ALS[END_REF]Turner et al., 1995;Turner & Weismer, 1993;Weismer et al., 2000, Turner & Weismer, 1993;[START_REF] Rong | Automatic extraction of abnormal lip movement features from the alternating motion rate task in amyotrophic lateral sclerosis[END_REF]Caruso & Burton, 1987;[START_REF] Kuruvilla-Dugdale | An investigation of clear speech effects on articulatory kinematics in talkers with ALS[END_REF]. Speech rate was shown to decline with the progression of the dysarthria [START_REF] Allison | Shorter Sentence Length Maximizes Intelligibility and Speech Motor Performance in Persons With Dysarthria Due to Amyotrophic Lateral Sclerosis[END_REF]Lee et al., 2018;Lee & Bell, 2018;[START_REF] Yunusova | Profiling Speech and Pausing in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)[END_REF][START_REF] Yunusova | Clinical Measures of Bulbar Dysfunction in ALS[END_REF].

Because dysarthria is presumably spastic only in PLS and flaccid only in SBMA, the correct distinction between spastic dysarthria, flaccid dysarthria and mixed spastic-flaccid dysarthria associated with ALS can assist neurologic differential diagnosis. The current study includes these three distinct groups to investigate whether a set of acoustic biomarkers differentiates among flaccid, spastic, and mixed spastic-flaccid dysarthria, associated with these three neurodegenerative MNDs.

Acoustic measures can be used as biomarkers of dysarthria in neurodegenerative diseases. A method using Mel Frequency Cesptral Coefficients (MFCCs), which provide a measure of spectral energy changes over time, recently showed to be a sensitive tool in capturing and identifying specificities in vocal tract shape changes from one articulatory configuration to the next, in flaccid-spastic dysarthria in ALS and hypokinetic dysarthria associated with Parkinson Disease [START_REF] Slis | Analysing spectral changes over time to identify articulatory impairments in dysarthria[END_REF]. MFCCs provide a measure of spectral energy in a temporal window; changes in these values from window to window reflect how this spectral energy changes over time. These spectral changes have shown to correlate with vocal tract shape modifications [START_REF] Goldstein | The Role of Temporal Modulation in Sensorimotor Interaction[END_REF]. [START_REF] Slis | Analysing spectral changes over time to identify articulatory impairments in dysarthria[END_REF] measured the spectral energy changes over time during the productions of successive identical vowel-glide syllables. These productions were characterized in terms of the overall amount of acoustic change in the sequence -which is expected to be large to reflect the acoustic contrast between the segments -, and in terms of variability of these acoustic changes -which is expected to be low to reflect the fact that the successive identical syllables are articulated the same way. Compared to controls and dysarthric patients with Parkinson Disease, dysarthric patients with ALS showed reduced values of these spectral changes over time, suggesting hypoarticulation, and more variability indicating instability in articulatory movements.

The goal of the current study was to capture spectral changes over time using MFCCs during the production of successive identical vowel-glide sequences to differentiate among the three dysarthria subtypes, associated with ALS, PLS and SBMA. Based on previous studies, we expected a smaller amount of spectral change, suggesting hypoarticulation, in speakers with ALS and PLS, and higher variability in speakers with ALS compared to controls, suggesting instability in articulatory movements. Due to the lack of research available for dysarthric speech of speakers diagnosed with PLS and SBMA, no specific hypotheses related to variability were formulated for these speakers. To further differentiate dysarthria subtypes, we also examined temporal measures during the production of the same sequences. Based on previous literature, slower sequence productions were expected in speakers with ALS and PLS compared to speakers with SBMA.

Finally, we explored the relation between the acoustic measures and dysarthria severity. For speakers with ALS, it was expected that the amount of the spectral changes correlated negatively with severity, and thus decreased, and duration of utterances correlated positively, and increased with the severity of dysarthria. Due to the lack of research available in dysarthric speech associated with PLS and SBMA, no specific hypotheses were formulated about the relation between the acoustic measures and dysarthria severity for speakers with these two pathologies.

Methods

Participants

One hundred and thirty-eight participants aged between 23 and 90 years, were enrolled in this experiment: seventy non-dysarthric French controls (forty from Paris and thirty from Geneva) who reported no history of neurological impairment or speech and language disorders and sixty-eight participants diagnosed with MNDs contributed speech recordings to this study. Among the patients, thirty-three patients with ALS, seventeen patients diagnosed with PLS and eighteen patients diagnosed with SBMA were recruited (see Table 1 for distribution of the groups). All patients were diagnosed by neurologists of the "ALS and other rare MNDs expert center" of the Pitié-Salpêtrière University Hospital (Paris, France). ALS patients were diagnosed based on the El Escorial criteria [START_REF] Brooks | El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis and Other Motor Neuron Disorders: Official Publication of the World Federation of Neurology[END_REF] and had "probable" or "definite" ALS. PLS patients were diagnosed based on the consensus diagnostic criteria and had "probable" or "definite" PLS [START_REF] Gordon | The natural history of primary lateral sclerosis[END_REF][START_REF] Turner | Primary lateral sclerosis: Consensus diagnostic criteria[END_REF]. SBMA patients were diagnosed based on the genetic test that estimates CAG expansion number in the androgen-receptor genes, considering more than 38 repeats to be pathologic. The protocol for the study (including participant recruitment, data management, consent forms and information leaflets) was approved by the following Ethics Committees: CPP Sud-Est III, ClinicalTrial.gov ID: NCT03560661 for the patients and the Commission Cantonale d'Ethique de la Recherche -2018-00212 (Switzerland) and CPP Sud Ouest et Outre Mer (ID_RCB: 2019-A02553-54) (France) for the control group recruitment. Prior to inclusion, all study participants provided informed consent.

To be eligible for this study, patients had to show presence of functional signs of bulbar involvement and be diagnosed with mild to moderate dysarthria. Subjects were ineligible for inclusion if they showed evidence of cognitive impairment and/or respiratory troubles according to the routine neurologist examination.

The presence of functional signs of bulbar involvement was assessed by an experienced SLP (first author) using the bulbar subscore of the ALSFRS-R scale [START_REF] Cedarbaum | The ALSFRS-R: A revised ALS functional rating scale that incorporates assessments of respiratory function. BDNF ALS Study Group (Phase III)[END_REF]. This subscore reflects the bulbar related handicap across three items (speech, salivation, and swallowing) that are rated on a 5-point scale (from 0 = severe impairment to 4 = normal function) yielding a maximum bulbar subscore of 12. A bulbar subscore less than 12 indicates impaired orofacial function.

The severity of dysarthria was assessed by the perceptive score (PS) from the "Batterie d'Evaluation Clinique de la Dysarthrie" (BECD) (Auzou & Rolland-Monnoury, 2019). The perceptive score (PS) is a composite score summing the evaluation of five speech dimensions (intelligibility, naturalness of speech, prosody, voice quality and articulatory precision) with a five-point scale score for each dimension (0 = normal, 1 = mild, 2 = moderate, 3 = severe and 4 = very severe impairment). A recent study investigating clinician-based adjectival rating of dysarthria severity in ALS found excellent intrarater and interrater reliability [START_REF] Stipancic | You Say Severe, I Say Mild": Toward an Empirical Classification of Dysarthria Severity[END_REF]. The total sum /20 indicates the severity of dysarthria following the cutoffs for severity group assignment suggested in the BECD: 1-6 = mild dysarthria and 7-13 = moderate dysarthria. Two experienced SLPs rated the dysarthria severity by listening to three-to-fiveminutes samples of connected speech. One of the SLPs (first author) recorded the patients and provided the severity ratings at the same time. The other SLP performed the ratings on the recordings of the same sample of connected speech. Since the first rater was not blind to the participants neurological deficits, the second rater was informed about the patients' diagnosis. Based on the level of overall score/20, intraclass correlations (ICCs) were used to calculate the PS score interrater reliability (as described in [START_REF] Koo | A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research[END_REF]. ICCs estimates and their 95% confidence intervals (CI) were calculated, based on a single rating, absoluteagreement, two-way random-effects model in R. Overall reliability between the two raters were excellent with a value greater than .90 (ICC = .94, p < .0001, 95%-CI: .91 < ICC < .96).

For ALS and PLS, reliability was excellent as well (ICC = .94, p < .0001, 95%-CI: .87 < ICC < .97 and ICC = .97, p < .0001, 95%-CI: .92 < ICC < .99 respectively). For SBMA, reliability was good (ICC = .89, p < .0001, 95%-CI: .72 < ICC < .96). Based on these high levels of interrater reliability, the PS scores were averaged. Table 1 presents the population. As shown by the ALSFRS-R bulbar subscore, the population was quite homogenous in terms of functional signs of bulbar involvement.

However, even though both mild and moderate cases of dysarthria were present in all subgroups (see regression Figure 3), speakers in the SBMA group showed milder scores for dysarthria overall which is also frequently observed in clinical practice. During recruitment, a first screening of the patients made by the first author permitted to determine the subtypes of dysarthria based on the cardinal speech features corresponding to each dysarthria subtype (Darley et al., 1969a).

Speech material and recording procedure

The speakers were instructed to produce three sequences composed of three repetitions of the same syllable, either a vowel-glide syllable in /ajajaj/ and /ujujuj/, both meaning "ouch, ouch, ouch" in French; or a glide-vowel syllable in /wiwiwi/ meaning "yes, yes, yes" in French. The stimuli were selected because they elicit successive vocal tract shape modifications which result in easily traceable acoustic outcomes with continuous resonances.

During /ajajaj/, the aperture of the vocal tract changes during the transition from vowel to glide with active tongue and/or jaw movements resulting in dominant resonance changes in the lower part of the spectrum (F1 area). /ujujuj/ and /wiwiwi/ require back/front movement of the tongue and successive rounding/unrounding of the lips, which induce acoustic changes dominant in mid frequency range. These last two sequences also vary in terms of syllabic structure with a nucleus-coda structure in /ujujuj/ and an onset-nucleus structure for /wiwiwi/.

The orthographic forms "aïe, aïe, aïe", "ouille, ouille, ouille" and "oui, oui, oui" were presented on the screen of a laptop and produced at a comfortable rate and loudness. Each sequence was repeated four times (n = 12 sequences per speaker) and sequences were presented in the same order for all speakers. The participants were instructed to produce each sequence in a continuous manner. Each participant attended a single recording session.

Recordings took place in a quiet room with the first author present. A high-quality acoustic recording equipment was used with a Focusrite Scarlett (2i4) external audiocard and a professional quality Shure SM35-XLR earset microphone that was positioned approximately 3 cm from the mouth during recordings. The speech was recorded at a sampling rate of 22.5 KHz and 16-bit resolution.

Acoustic data analysis

All sound files were resampled to 16 KHz. Audio based segmentation and annotation of each sequence were manually performed in Praat (Praat: Doing Phonetics by Computer, 2020). To compute the amount of acoustic change produced over the utterance Mel Frequency Cepstral Coefficients (MFCCs) were calculated as follows. First, the spectra were calculated over 25 ms time frames sliding over time in 2 ms steps. To target the frequencies relevant for vocal tract shape changes, only the spectral coefficients between 300 and 8000 Hz were considered. The trimmed frequency scale was then converted into a Mel frequency scale, which approaches the filter characteristics of the human ear [START_REF] O'shaughnessy | Speech Communications: Human and Machine[END_REF].

Next, the spectra were log-transformed and submitted to a Discrete Cosine Transformation (Davis & Mermelstein, 1980), resulting in a set of coefficients. Because the 1 st coefficient contains information about the overall average spectral energy, this coefficient was disregarded and only the 2 nd to 13 th coefficient were considered. Next the differences between coefficients 2-13 in two consecutive windows were calculated and squared; information about the direction of change is thus removed and only the magnitude of energy change is preserved. Finally, these difference values were summed (see also [START_REF] Slis | Analysing spectral changes over time to identify articulatory impairments in dysarthria[END_REF]. This method results in values of total squared changes of mel frequency cepstral coefficients, referred to as TSC_MFCCs. Plotting all TSC_MFCCs, a contour resulted, consisting of increasing and decreasing TSC_MFCC values as illustrated in Figure 1, which presents the contours of the degree of acoustic change during a /ujujuj/ sequence, produced four times by a talker of each group.

Maxima in this contour aligns with the part of the speech production, during which the most vocal tract shape change is observed, i.e., during the transition from a vowel to a glide or glide to a vowel. Minima align with the part during which the vocal tract shape is relatively steady, i.e., during the steady state of the speech production.

Derived metrics

The complete TSC_MFCC contour was delimited from the steady part of the first segment (i.e., the first valley) to the steady part of the last segment (i.e., the last valley). Next, three measures were extracted (see also [START_REF] Slis | Analysing spectral changes over time to identify articulatory impairments in dysarthria[END_REF] : -VARCO: the coefficient of variation of the complete contour determined the degree of variability of the acoustic changes over the sequence. In other words, it aimed to capture the stability in the acoustic contrasts, resulting from repeated successive syllables of the sequence. The value is expected to be lower if the successive elements of the sequence are articulated the same way and to increase with instability in the articulation over the successive elements.

-eventDUR: to capture differences in articulation rate, maximum and minimum velocity values in the contour were automatically determined by a peak-picking algorithm in MATLAB. Values were then manually checked. Each interval duration from each valley to the next was then computed. These intervals included the acoustic transition between one segment to the next in the /ajajaj/, /ujujuj/, /wiwiwi/ sequences since valleys in the TSC_MFCC contour occur where there is little acoustic changes before and after each transition.

Statistical analysis

Main effects of SPEAKER CONDITION (CONTROLS, ALS, PLS and SBMA) and SEQUENCE TYPE (/aj/, /uj/, /wi/) as well as the interaction between the two were examined, using linear mixed effect models (LMM) for the three acoustic metrics (MEAN, VARCO, eventDUR). Because measurements were based on 4 repetitions of a sequence for each speaker, speakers were considered as random effect variables. When findings of the LMM models revealed statistically main or interaction effects, post hoc comparisons were performed applying Tukey adjustments for multiples comparisons. The critical alpha level for all tests was set to p < .05.

To assess the possible relationship between the dysarthria severity and the dependent variables, individual linear regressions for each pathological condition (ALS, PLS, SBMA)

were used. Since the perceptual score is measured by participant and is the same for the 12 sequences (3 sequences * 4 repetitions) for each participant, we decided to create a unique variable by combining the values of the 12 sequences to avoid convergence issues. Hence, speakers were not considered as random effect variables. Because normality and/or homogeneity assumptions were frequently rejected, we did not consider standard error and t-values which can be biased in such cases with potential misleading p values. Confidence intervals for the linear regression slopes, estimated by using a bootstrap approach, were used to test the null hypothesis. The linear association between the dysarthria severity level and the dependent variables was considered statistically significant when the 95% confidence interval excluded the value of 0.

For each analysis, values below the first percentile and over the 99th percentile were removed for each dependent variable. Residuals normality assumptions were checked using Shapiro-Wilk test and quantile-quantile plots. The residual homogeneity assumption was checked visually by plotting the standardized residuals as a function of the corresponding fitted values. Because data were unbalanced and there was an interaction effect between the independent variables, a type III sum of squares methodology was used to assess significance, and a p-value of <.05 for two-sided test was considered significant.

All statistical analyses were done using R (version 4.0.2). Linear mixed effect models were fitted using the lmer function of the lme4 [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF] and the lmerTest packages [START_REF] Kuznetsova | lmerTest Package: Tests in Linear Mixed Effects Models[END_REF]. P values were computed using Kenward-Roger 's approximation of the degree of freedom. Post-hoc comparisons were done using the emmeans function of the emmeans package [START_REF] Lenth | emmeans: Estimated Marginal Means, aka Least-Squares Means[END_REF], and confidence intervals of slope were estimated using the Boot function of the car package [START_REF] Fox | An {R} Companion to Applied Regression, Third Edition[END_REF].

Results

SPEAKER CONDITION (CONTROL 1 , ALS, PLS and SBMA) and SEQUENCE TYPE Figure 2 presents the condition averages for the three measures MEAN, VARCO and eventDUR across all sequences.

i. MEAN

A significant main effect was found for SPEAKER CONDITION and SEQUENCE TYPE (see Table 2). Overall, MEAN acoustic change was significantly smaller for ALS and PLS compared with SBMA (respectively p < .001 and p < .01) and controls (p < .001). No significant difference was found between ALS and PLS (p = .5) and between SBMA and controls (p = .1).

Overall, the sequence /ajajaj/ elicited significantly smaller MEAN acoustic change than /ujujuj/ and /wiwiwi/ (p < .0001). No significant difference was found between /ujujuj/ and /wiwiwi/ (p = .3).

The interaction between SPEAKER CONDITION x SEQUENCE TYPE was significant. During the production of /ajajaj/, the MEAN acoustic change for ALS speakers was significantly smaller than for CONTROL and SBMA speakers, and the MEAN acoustic change for PLS speakers was significantly smaller compared to SBMA speakers (see Table 3). No significant difference was found between ALS and PLS, CONTROL and PLS, and CONTROL and SBMA (ALS < SBMA & controls; PLS < SBMA).

During the production of /ujujuj/and /wiwiwi/, the MEAN acoustic change for ALS and PLS speakers was significantly smaller than for CONTROL and SBMA and the MEAN acoustic change of SBMA speakers was significantly smaller than that of CONTROL (ALS & PLS < SBMA < CONTROL). No significant difference was found between the two conditions ALS and PLS.

As shown in Table 4, all the groups showed a larger degree of acoustic change over the backfront/rounded-unrounded sequences compared to /ajajaj/. Only for speakers with PLS, the sequence /wiwiwi/ elicited significantly greater MEAN acoustic change than the sequence /ujujuj/.

ii. VARCO

A main effect was found for SPEAKER CONDITION and SEQUENCE TYPE (see Table 2).

ALS showed higher VARCO values than CONTROL (p < .0001), PLS (p = .04) and SBMA (p = .001). The sequences /ujujuj/ and /wiwiwi/ resulted in greater VARCO than the sequence /ajajaj/ (p < .0001).

The SPEAKER CONDITION x SEQUENCE TYPE interaction was significant. As shown in Table 5, a significantly greater VARCO in the ALS condition compared to CONTROL across the three sequences /ajajaj/, /ujujuj/ and /wiwiwi/ was observed. During the /ujujuj/ production, ALS speakers also showed greater VARCO compared to the PLS and the SBMA speakers, and PLS speakers had significantly greater VARCO than CONTROL speakers (ALS > PLS & SBMA; PLS > CONTROL). Finally, during the production of the sequence /wiwiwi/, ALS speakers also showed greater VARCO compared to the SBMA.

As shown in Table 6, for both PLS and ALS, the alternating back-front/rounded-unrounded sequences induced more irregularity in the successive articulation that the open-close /ajajaj/. It is shown in the VARCO for /ujujuj/ and /wiwiwi/ for ALS and for /ujujuj/ in PLS.

In SBMA, no difference in VARCO is found according to the sequence.

iii. eventDUR

A significant effect was found for SPEAKER CONDITION, for SPEAKER CONDITION x SEQUENCE TYPE interaction, but not for SEQUENCE TYPE (see Table 2). As shown in Table 7 and8, ALS and PLS speakers are slower than both SBMA and CONTROL speakers for all sequences. Again, the back-front/rounded-unrounded sequences differed from the /ajajaj/ sequence for the ALS speakers, with longer eventDUR for /ajajaj/ compared with /ujujuj/ and /wiwiwi/. For speakers with PLS, the /wiwiwi/ sequence showed longer eventDUR values than /ajajaj/. For CONTROL and speakers with SBMA, eventDUR values did not change according to SEQUENCE TYPE.

Relationship between metrics and dysarthria severity

Relations between the acoustic measurements and dysarthria severity (expressed in a perceptual score taken as a discrete quantitative variable) are shown in Figure 3. Table 9 displays the findings of the linear regression analyses for each pathological condition.

The linear association between dysarthria severity and MEAN was statistically significant only in ALS and PLS. MEAN acoustic change decreases when dysarthria severity increases. The linear association between the dysarthria severity and VARCO was statistically significant only in SBMA. As shown in Figure 3, there was an apparent decrease in VARCO with dysarthria severity in SBMA. However, this trend was mainly due to sparse distribution of the mild dysarthric speakers (PS < 5), with some speakers showing a high VARCO, similar to that of ALS speakers. No linear association was found between dysarthria severity and eventDUR in any condition. In ALS and PLS, three clusters appear in the distribution (Figure 3): mild dysarthric speakers (PS < 7) showed eventDUR around 50-65 ms, moderate dysarthric speakers (7 < PS < 11) showed longer eventDUR between 65-85 ms with a few speakers between 70 and 85 ms, but a few speakers with more severe dysarthria (11 < PS < 13) showed eventDUR that were shorter (< 70 ms) than the moderate dysarthric speakers.

Discussion

The current study explored the use of an acoustic measure, which indirectly captured vocal tract shape changes, to differentiate flaccid, spastic, and mixed spastic flaccid dysarthria subtypes associated with SBMA, PLS and ALS respectively. Several findings indicate that the tool is efficient in describing articulatory differences between the three dysarthria subtypes and the following sections discuss for each dysarthria subtype how the results of mean acoustic change, variability and duration inform on these distinctions.

Flaccid dysarthria in SBMA

For flaccid dysarthria in SBMA, involving LMN system, it was hypothesized that temporal aspects of speech were reduced compared to controls as a consequence of motor weakness or as a strategy to compensate for this weakness [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF]. Studying articulatory impairments in SBMA was novel in this research, as they have already been reported in LMN involvement due to other origins before [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF]. No hypotheses were formulated concerning the amount and the variability of the acoustic change, as well as the relation between dysarthria severity and the outcome measures.

Mean acoustic change

Compared to speech of non-dysarthric speakers, the speech of patients with SBMA showed a lower amount of acoustic energy change, suggesting diminished possibilities to change the vocal tract shape. As stated earlier, the acoustic changes, measured by the MFCCs, capture changes between the successive vocal tract configurations required to produce the three-syllable sequences. A reduced amount of acoustic energy change was thus interpreted as a sign of hypoarticulation during the vowel glide and glide vowel sequences. Interestingly, signs of hypoarticulation were only found in the productions of the sequences/ujujuj/ and /wiwiwi/. These sequences specifically involve back-front tongue movements and roundedunrounded lip movements. The marked amyotrophy and weakness of the tongue described in SBMA, causing swallowing problems [START_REF] Banno | Swallowing markers in spinal and bulbar muscular atrophy[END_REF][START_REF] Mano | Tongue pressure as a novel biomarker of spinal and bulbar muscular atrophy[END_REF] possibly explain why the alternating front-back movements of the tongue required for these sequences were difficult to achieve for the speakers with SBMA. Despite the reported jaw weakness in SBMA resulting in cases of jaw dropping [START_REF] Larsen | Jaw drop" as an atypical manifestation of Kennedy's disease[END_REF][START_REF] Sumner | Jaw drop in Kennedy's disease[END_REF], speakers in the current study managed to preserve acoustic spectral energy changes during /ajajaj/. The observation that speakers with SBMA showed a tendency for larger values of acoustic energy change than the other groups during productions requiring jaw movements (Figure 2), suggests a jaw aperture overshoot during /ajajaj/ resulting from poor control; this interpretation is merely speculative and must be considered as an interesting line of future research, exploring the behavior of the jaw in speakers with SMBA.

The amount of acoustic change did not decrease with increased perceptually assessed dysarthria severity for this group of speakers with pure flaccid dysarthria. Compared to the other populations, there was a large diversity in speaker specific patterns, as shown in Figure 3, which was not related to severity. Different possible explanations for this result are proposed. First, the findings might have been influenced by the way severity was assessed.

The Perceptual Score indicated a composite measure of severity, not only related to articulatory dimensions. Therefore, the severity of dysarthria might have been rated on other prominent altered speech dimensions, such as voice quality or nasal resonance, which are particularly affected in SBMA [START_REF] Tanaka | Distinct acoustic features in spinal and bulbar muscular atrophy patients with laryngospasm[END_REF][START_REF] Tanaka | Nasometric Scores in spinal and bulbar muscular atrophy-Effects of palatal lift prosthesis on dysarthria and dysphagia[END_REF]. Second, it is speculated that the decrease of acoustic change was not affected by severity progression in this population because our sample did not cover the same range of severity as the other populations. Indeed, SBMA patients usually present mild levels of dysarthria and this was reflected in our population; only 5 moderate (vs. 13 mild) dysarthric patients were recruited.

Variability

Poor lingual mobility did not seem to be related to the stability of vocal tract shape changes. Indeed, except for a few speakers, SBMA speakers showed similar low variability values as controls and PLS speakers. Recall that the VARCO measure captures the articulatory stability of the successive syllables in the sequence. It was speculated that one of the reasons for this stability was due to the slow progression of this disease, allowing patients to find compensatory adjustments. This idea was supported by the high VARCO profiles of some of the mildest dysarthric SBMA speakers, while all speakers with more progression in the dysarthria (PS>5) achieved to produce more stable acoustic patterns (Figure 3).

Duration

Our findings do not support the notion that tongue weakness results in slow speech rate in flaccid dysarthria associated with SBMA as suggested in the literature for flaccid dysarthria due to other etiologies [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF]. Speakers with pure flaccid dysarthria in SBMA showed the same temporal patterns as controls in all sequences possibly related to the fact that the group of SBMA patients were mildly dysarthric (PS<7).

Spastic and spastic-flaccid dysarthria in PLS and ALS

Mean acoustic change

Based on previous studies stated earlier on articulatory impairments in ALS and other pathologies involving pure UMN dysfunction, it was expected that speakers with ALS and PLS showed a reduction in the degree of acoustic energy changes, implicating smaller vocal tract shape changes and thus hypoarticulation. This was indeed the case: speakers with dysarthria associated with ALS and PLS both displayed a comparable low amount of acoustic change, which was even lower than observed for speakers with SBMA. For both groups the acoustic energy differences declined with dysarthria severity, and confirmed the findings on progressively reduced tongue movement range in speakers with ALS [START_REF] Kuruvilla | Spatiotemporal coupling of the tongue in amyotrophic lateral sclerosis[END_REF]Lee et al., 2018;Lee & Bell, 2018).

Compared to flaccid dysarthric speakers and non-dysarthric speakers, the back-front sequences /ujujuj/ and /wiwiwi/ were especially affected, which is in agreement with the previous kinematic studies demonstrating a reduced articulatory range of tongue movements detected in the anterior-posterior dimension in ALS patients (Lee & Bell, 2018). Compared to speakers with pure flaccid dysarthria associated with SBMA, however, both speakers with mixed spastic-flaccid and pure spastic dysarthria also displayed significant reduced acoustic energy changes in the sequence /ajajaj/. It is speculated that the absence of hypoarticulation in the /ajajaj/ sequence for the SBMA speakers is caused by the fact that for this sequence, compensation is still possible to achieve the appropriate opening and closing movements.

Muscle weakness associated with LMN involvement only affects isolated muscles but the spasticity typically associated with UMN involvement affects patterns of movements and coordination between them [START_REF] Duffy | Motor speech disorders e-book: Substrates, differential diagnosis, and management[END_REF]. Impaired movement patterns and coordination probably explain the lower values of mean energy change in dysarthric speakers with UMN involvement. It is speculated that the similarities, found between dysarthric speakers associated with ALS and PLS, are originating as a consequence of their shared UMN involvement. Although studies have shown that the jaw displayed exaggerated range of movement in ALS [START_REF] Kuruvilla | Spatiotemporal coupling of the tongue in amyotrophic lateral sclerosis[END_REF]Lee et al., 2018[START_REF] Lee | Direction-Specific Jaw Dysfunction and Its Impact on Tongue Movement in Individuals With Dysarthria Secondary to Amyotrophic Lateral Sclerosis[END_REF]Lee & Bell, 2018;[START_REF] Yunusova | Articulatory Movements During Vowels in Speakers With Dysarthria and Healthy Controls[END_REF], it seemed that this compensation was not achieved by the ALS and PLS speakers in this study. A possible interpretation explaining the lack of compensation by ALS and PLS speakers, is that these speakers may present jaw spasticity resulting in reduced range of jaw movements (for a review on spasticity, see [START_REF] Synnot | Interventions for managing skeletal muscle spasticity following traumatic brain injury[END_REF].

Variability

Whereas speakers with spastic and mixed dysarthria set themselves apart from speakers with flaccid dysarthria and non-dysarthric speakers regarding the amount of acoustic energy change, they can be separated from each other further in terms of variability of acoustic energy change. While similar patterns of mean acoustic energy change was shown in speakers with spastic and mixed dysarthria, speakers with mixed dysarthria showed a much higher degree of variability overall, especially caused by the /ujujuj/ sequence. This great variability observed in patients with ALS is likely multifactorial. First, the well-known fatigue in ALS [START_REF] Beukelman | Communication Support for People with ALS[END_REF] might have contributed to higher variability and second, the fact that the progression of this disease is very fast compared with PLS, prevents speakers from establishing solid compensatory strategies, likely resulting in more variability.

Finally, because both the UMN and LMN systems are affected in ALS resulting in mixed dysarthria, we speculate that productive compensation strategies are more difficult to achieve.

Duration

Based on the literature cited in introduction which demonstrated impaired temporal aspects in speech production, in ALS as well as in other pathologies involving pure UMN dysfunction, we expected slower sequence production in mixed and spastic dysarthria. Our results indeed confirmed that both spastic and mixed dysarthric speakers showed longer segment duration in the vowel-glide sequences than the controls and speakers with flaccid dysarthria. These results are comparable to those of [START_REF] Mefferd | Articulatory-to-Acoustic Relations in Talkers With Dysarthria: A First Analysis[END_REF] and Lee et al. (2018), who found respectively that the duration of the diphthong /ai/ in the word "kite", and /wi/ in the word "whip" was prolonged for speakers with mixed dysarthria. Interestingly, the current findings showed an interaction in the sense that the duration values for /wiwiwi/ were higher than /ajajaj/ for the spastic dysarthric speakers while the opposite was found for the mixed dysarthric speakers (/ajajaj/>/wiwiwi/). It is speculated that the slower duration of the sequence /ajajaj/ for speakers associated with ALS was caused by the fact that the jaw is especially affected in this disease, maybe because both UMN and LMN are involved. The exact reason for this finding, however, is not clear and the authors cannot provide a satisfactory explanation for this interaction.

Both in ALS and PLS, speakers with more severe dysarthria showed a slower speech rate than speakers with milder dysarthria. The lack of association, however, between the temporal aspects and the severity in this study is contradictory to our expectation. Indeed, a link between severity and slow rate has been described in the literature, particularly in ALS (for instance [START_REF] Allison | Shorter Sentence Length Maximizes Intelligibility and Speech Motor Performance in Persons With Dysarthria Due to Amyotrophic Lateral Sclerosis[END_REF]Lee et al., 2018;Lee & Bell, 2018). A possible explanation of the lack of association in this study is that 8 patients with ALS and PLS, judged with more severe dysarthria (11<PS<13), demonstrated shorter eventDUR than patients who were judged less dysarthric (7<PS<10). Further, one patient with ALS judged with mild dysarthria, showed the longest eventDUR. While speech rate and intelligibility are usually employed as severity indices in ALS (Ball et al., 2002;[START_REF] Green | Bulbar and speech motor assessment in ALS: Challenges and future directions[END_REF][START_REF] Kuruvilla | Spatiotemporal coupling of the tongue in amyotrophic lateral sclerosis[END_REF]Lee et al., 2018), we used a generalized rating of speech with five dimensions of speech (intelligibility, prosody, voice quality, naturalness and articulatory precision) making speech rate less important as a determining factor. It is possible that the severity of dysarthria of each patient was judged according to different dimensions, not specifically related to temporal aspects.

Limitation of the study

One of the factors that complicates studies like the current one is that the rate of disease progression varies considerably across these diseases. ALS can be a very fast progressive disease, leaving little time to develop compensation strategies, whereas PLS and SBMA are typically very slow in the speed of progression. Since the dysarthria characteristics may not only be related to UMN or LMN involvement, but also to the presence or absence of compensation strategies, our findings need to be considered with caution. In addition, our findings regarding the relation between severity and the outcome measures studied here likely differ from studies using severity indices such as speech rate or intelligibility. Finally, more balanced sample size across dysarthria severity continuum in each subtype would provide stronger conclusions about the relation between severity and the outcome measures.

Clinical implications and conclusion

The acoustic parameters used in this study partially differentiated among the dysarthria subtypes in relation to MNDs: smaller vocal tract shape changes from one articulatory configuration to the next were shown in flaccid dysarthria but to a less extent than in spastic and spastic-flaccid dysarthria. These latter showed similar acoustic patterns.

We speculate that these similarities are due to their common spastic component. Yet, crucial differences between spastic and spastic-flaccid dysarthria were found in the stability of the acoustic patterns. In spastic-flaccid dysarthria, these patterns were much more variable, suggesting articulatory instability of the successive syllables in the sequence. The method used in the current study forms a promising clinical tool as a diagnostic marker of diminished vocal tract shape changes, interpreted as hypoarticulation here, even at mild stage of dysarthria progression, and as an objective monitoring indicator of the disease progression in the bulbar territory in ALS and PLS. Though, future research goals which are interesting to pursue are investigating more in depth the relation between the current acoustic measures and articulatory patterns to better understand articulatory deficiencies in MNDs. Because this method does not require advanced instrumentation, the acoustic measures used in this study are well-suited to assess vocal tract shape changes in a clinical setting. We are currently working on integrating this method in the MonPaGe assessment tool [START_REF] Laganaro | Sensitivity and specificity of an acoustic-and perceptual-based tool for assessing motor speech disorders in French: The MonPaGe-screening protocol[END_REF][START_REF] Pernon | MonPaGe, un outil de screening francophone informatisé d'évaluation perceptive et acoustique des troubles moteurs de la parole (dysarthries, apraxie de la parole[END_REF].

-

  MEAN: the mean of the complete TSC_MFCC values capturing the average amount of acoustic change over time during the sequence. It is expected to lower with decreased acoustic contrastiveness between the successive segments, for example during a hypoarticulated sequence with little vocal tract changes.

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 MEAN, VARCO and eventDUR as a function of sequences. Dispersion bars around the mean values represent confidence intervals.

Figure 3

 3 Figure 3 Relationships between the three acoustic measures (MEAN, VARCO, eventDUR) and the dysarthria severity (indexed by the perceptual score taken as a discrete quantitative variable) for the three dysarthric populations. Shaded areas indicate 95% confidence interval of regression estimates. R 2 is the coefficient of determination.

Table 1 Cohort

 1

	Group	N	Age	ALSFRS-R	Perceptive Score	Dysarthria subtypes
		(M/F)	Mean (SD) [Range]	Bulbar Mean (SD) Subscore	(BECD) Mean (SD)
	ALS	20/13	63.67 (10.90)	9.00 (1.32)	8.05 (3.11)	Mixed flaccid-spastic
			[36-86]
	PLS	10/7	67.18 (8.18)	8.94 (1.25)	7.65 (3.56)	Spastic
			[55-88]
	SBMA	18/0 2	63.56 (12.28)	9.33 (1.19)	5.29 (2.75)	Flaccid
			[43-85]
	CONTROLS	29/41	58.10 (20.02)	NA	NA	NA
			[23-90]

description in terms of distribution across sex ( N(M/F): number of male and female speakers), age (mean, standard deviation (SD) and range), ALSFRS bulbar subscores, used to assess bulbar involvement (mean and standard deviation), PS scores, used t o assess the dysarthria severity (mean and standard deviation) and dysarthria subtypes. Note. ALSFRS-R: ALS Functional Rating Scale -Revised; BECD: Batterie d'Evaluation Clinique de la Dysarthrie.

2 SBMA is a male, X-linked recessive disease

Table 3

 3 Estimate, standard error (SE), t and p values for condition's comparisons for the metric M E A N acoustic change.

	MEAN

Table 4

 4 Estimate, SE, t and p values for the sequence's comparisons for the metric

	M E A N .

Table 5

 5 Estimate, SE, t and p values for the condition's comparisons for the metricV A R C O .

	42

Table 6

 6 Estimate, SE, t and p values for the sequence's comparisons for the metric

	V A R C O .

Table 7

 7 Estimate, SE, t and p values for the condition's comparisons for the metric eventDUR.Table8 Estimate, SE, t and p values for the sequence's comparisons for the metric eventDUR.

	44

Variables were compared between the controls from Geneva and from Paris with the talker's origin as fixed effect. No statistical differences were found between them, excluding a potential regional differences effect. They were thus grouped in a single control population in the analyses.

Acknowledgments

This research was funded by Assistance Publique -Hôpitaux de Paris, Délégation de la Recherche Clinique et de l'Innovation (DRCI) (awarded to the first author) and by the Swiss National Science Foundation, Sinergia program (CRSII5_173711, 10.2017-9.2020). The authors are thankful to the participants for taking part in this project. The authors would also like to thank François Salachas, Nadine Le Forestier, Pierre-François Pradat, Timothée Lenglet, Maria Del Mar Amador, Adèle Hesters, Giorgia Querin for supporting patients' recruitment and Michaela Pernon, Claire Della Vedova and Speech'n Co colleagues for their help in this project. We thank the reviewers of the manuscript for their inspiring comments.

The authors declare no conflict of interest. This research was funded by Assistance Publique -Hôpitaux de Paris, Délégation de la Recherche Clinique et de l'Innovation (DRCI) (awarded to the first author) and by the Swiss National Science Foundation, Sinergia program (CRSII5_173711, 10.2017-9.2020).


Kim-Anh Nguyen-Peyre

Weiqiang Liu

Lazaros Papamanolis

Christian Kassasaya

Nour Bekeziz

Valentin Amar

Gabriel Nahas

Saskia Eckert

Pierre Vedel

Laurène Lenoir

Katy Drémon

Sabine Cléophax

France Pirenne

Frédéric Segonds

Suzanne Verlhac

Irène Vignon-Clementel ¥

Pablo Bartolucci

Impact of Carotid Geometry on Sickle Cell Diseases-related Cerebral Vasculopathy Development

Introduction

Sickle cell disease (SCD) is the most prevalent monogenic disorder resulting from a mutation in the -globin gene. Among its severe and multi-system complications, the cerebral vasculopathy (CV) generally occurring during childhood, is responsible for ischemic stroke, making SCD the first etiology of stroke in children and young adults (Guerriero RM, 2019, Ohene-Frempong K 1998). SCD-related overt strokes are typically due to large-artery stenosis of carotid siphon. This stenosis is preceded by a high intracranial arterial velocity on transcranial Doppler (> 200 cm/s) (Adams RJ 1992). We hypothesis that the high flow rate and carotid geometry in small SCD children could lead to pathological velocities apparition and promote platelet secretion of pro-fibrotic factors. Thus, a 3D patientspecific vessel model enables to study the physiopathology of CV development. In this study, by developing an in sillico and in vitro personalized 3D carotid model we aim to evaluate impact of carotid geometry on the apparition of pathological intracranial velocities and the platelet secretion of pro-fibrotic factors.

Materials and methods

Mathematical modelling of internal carotid and vascular flow

The internal carotid artery (ICA) with its terminal branches, anterior cerebral arterial (ACA) and middle cerebral arterial (MCA), was segmented from magnetic resonance angiography images for 4 SCD children < 5 years old (UF), 3 SCD children > 5 years old (OF) and 1 SCD adult (AD) on both sides. Blood flow was modelled using the 3D Navier-Stokes equations, assuming that blood is an incompressible Newtonian fluid. Pulsatile flow was assigned as an inlet boundary condition with mean flow rates varying from 5 to 15 mL/s which reflect the range of flow rates observed in SCD patients. TMMV and Dean number were calculated over five regions of interest, as shown in Fig 1A .  Segmented internal carotid arteries of an UF and an AD SCD patient were printed using stereolithography 3D printing.

Flow experiments in 3D printed carotid with platelet concentrates

Fresh apheresis platelet concentrates (APC) from 5 healthy donors were perfused in flow system containing either 3D-printed UF or AD carotid at either low or high flow (8 ml/s or 16 ml/s respectively) for 30 minutes. Platelet secretion of soluble CD62P (sCD62P), TGF-, and TSP-1 at 5 min, 15 and 30 min in the supernatants was measured by ELISA.

Results and discussions

Mathematical modelling of internal carotid and vascular blood flow

Simulation results show that for any inlet flow rate from 5 to 15 ml/s, the TMMV calculated in younger SCD children tend to be higher than in older patients. Consequently, most UF patients reach pathological TMMV (>200 m/s) at lower inlet flow rates compared to older patients (Fig 1B)

Platelet secretion of soluble factors in 3D-printed carotid

The high flow induces an important platelet secretion of the TGF-, a pro-fibrotic factor with increasing trend in UF than in AD carotid and in a time dependent manner (Fig 1D). In contrast, compared to resting condition, a similar sCD62P level is observed in either AD or UF carotid at low and high flow suggesting the absence of platelet activation in flow conditions, leading to a negligible platelet release of TSP-1, an anti-angiogenic and major endogenous activator of TGF-β. These results suggest that UF carotid geometry could promote platelet secretion of TGF which is accentuated by high TMMV independently with platelet activation.

Conclusions and perspectives

This study performed in a 3D personalized carotid model has demonstrated that at high flow which are generally observed in UF SCD patients, these patients are more likely to reach the pathological intracranial arterial velocities and the platelet secretion of pro-fibrotic factors compared to AD patients, leading to their high risk of stroke. The measurement of other platelet factors and the evaluation of geometrical features such as diameter, tortuosity and curvature in stenosis formation are in progress. These results could allow to explain the CV development in SCD. Fresh apheresis platelet concentrates (APC) from 5 healthy donors were perfused in flow system containing either 3D-printed AD or UF carotid at either low or high flow (8 ml/s or 16 ml/s respectively) for 30 minutes. Platelet secretion of TGFb, soluble CD62P and TSP at 5 min, 15 and 30 min in the supernatants was measured by ELISA.

The fold changes of each factor are computed as the ratio of concentration between the indicated and the resting condition (before perfusion). Data from 5 independent experiments. Bars represent the mean ± SD. The differences are not significant (Wilcoxon matched-pairs signed rank test p>0.05)

  . The pathological TMMV seem to appear firstly and mostly in the carotid siphon or post-siphon regions (Fig 1B). TMMV shows significant correlation with Dean number (R = 0.72, p-value= 1e-14), as shown in Fig 1C, indicating high flow rate, large curvature and small cross-sectional area of cerebral arteries are closely linked with high TMMV.

Figure 1 :

 1 Figure 1: Experiments in 3D personalized carotid model (A-C. In sillico model; D. 3D-printed carotid model) A. 3D personalized model with its flow inlet and 5 regions of interest (darker grey) for calculation of TMMV and Dean number. B. TMMV calculated in 8 patients (4 UF, 3 OF and 1 AD) over five regions of interest: Patient 1-8 is sorted by age: 3y2m, 4y1m, 4y2m, 4y8m, 5y5m, 7y11m, 11y3m and 23y respectively. C. Correlation between TMMV and Dean number for all patients for a mean flow rate of 5 mL/s. D. Platelet secretion of TGFb, soluble CD62P and TSP-1 in 3D-printed carotid models under flow condition.Fresh apheresis platelet concentrates (APC) from 5 healthy donors were perfused in flow system containing either 3D-printed AD or UF carotid at either low or high flow (8 ml/s or 16 ml/s respectively) for 30 minutes. Platelet secretion of TGFb, soluble CD62P and TSP at 5 min, 15 and 30 min in the supernatants was measured by ELISA.The fold changes of each factor are computed as the ratio of concentration between the indicated and the resting condition (before perfusion). Data from 5 independent experiments. Bars represent the mean ± SD. The differences are not significant (Wilcoxon matched-pairs signed rank test p>0.05)