Syringeal vocal folds do not have a voice in zebra finch vocal development.

Vocal behavior can be dramatically changed by both neural circuit development and postnatal maturation of the body. During song learning in songbirds, both the song system and syringeal muscles are functionally changing, but it is unknown if maturation of sound generators within the syrinx contributes to vocal development. Here we densely sample the respiratory pressure control space of the zebra finch syrinx in vitro. We show that the syrinx produces sound very efficiently and that key acoustic parameters, minimal fundamental frequency, entropy and source level, do not change over development in both sexes. Thus, our data suggest that the observed acoustic changes in vocal development must be attributed to changes in the motor control pathway, from song system circuitry to muscle force, and not by material property changes in the avian analog of the vocal folds. We propose that in songbirds, muscle use and training driven by the sexually dimorphic song system are the crucial drivers that lead to sexual dimorphism of the syringeal skeleton and musculature. The size and properties of the instrument are thus not changing, while its player is.

Evaluation of the Human Vocal Fold Lamina Propria Development Using Optical Coherence Tomography.

OBJECTIVES/HYPOTHESIS: Identifying distinctive features of the vocal fold (VF) during development could have significant clinical implications for treating voice disorders. This study investigates the structural organization of the VF microanatomy across gender and age groups using optical coherence tomography (OCT). STUDY DESIGN: Prospective clinical trial. MATERIALS AND METHODS: In vivo OCT images were acquired from 97 patients (58 males and 39 females) aged between 6 weeks and 27 years. All patients showed no signs of vocal fold pathology on endoscopy. Morphological features were extracted from OCT images and statistically compared between age groups. This study was performed at Massachusetts Eye and Ear between 2017 and 2019. RESULTS: All OCT acquisitions show a stratified microanatomy across age groups, even in newborns suggesting the presence of a superficial lamina propria (SLP) at birth. Furthermore, the optical scattering in the VF lamina propria changes according to age, suggesting subepithelial maturation. Although the epithelium thickness was relatively constant across age groups, the SLP showed a significant linear relationship between age and thickness (P = .016). Furthermore, a significant difference (P = .002) in SLP thickness was found between young adult males and females. The overall thickness of the entire mucosa did not change significantly with age. CONCLUSION: OCT is a noninvasive imaging modality capable of providing quantitative morphological features to describe the VF development. A stratified structure can be observed in OCT from newborns to young adults. Further investigations could combine OCT, acoustic measurements, and molecular sensitive techniques to provide a complete interpretation of the VF development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2558-E2565, 2021.

Mechanisms of larynx and vocal fold development and pathogenesis.

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.

Vocal state change through laryngeal development.

Across vertebrates, progressive changes in vocal behavior during postnatal development are typically attributed solely to developing neural circuits. How the changing body influences vocal development remains unknown. Here we show that state changes in the contact vocalizations of infant marmoset monkeys, which transition from noisy, low frequency cries to tonal, higher pitched vocalizations in adults, are caused partially by laryngeal development. Combining analyses of natural vocalizations, motorized excised larynx experiments, tensile material tests and high-speed imaging, we show that vocal state transition occurs via a sound source switch from vocal folds to apical vocal membranes, producing louder vocalizations with higher efficiency. We show with an empirically based model of descending motor control how neural circuits could interact with changing laryngeal dynamics, leading to adaptive vocal development. Our results emphasize the importance of embodied approaches to vocal development, where exploiting biomechanical consequences of changing material properties can simplify motor control, reducing the computational load on the developing brain.

Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure.

Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells' limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

Heterogeneity of Stem Cells in the Human Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal, and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

Registers in Infant Phonation.

The primary vocal registers of modal, falsetto, and fry have been studied in adults but not per se in infancy. The vocal ligament is thought to play a critical role in the modal-falsetto contrast but is still developing during infancy (Tateya and Tateya, 2015).41 Cover tissues are also implicated in the modal-fry contrast, but the low fundamental frequency (fo) cutoff of 70 Hz, shared between genders, suggests a psychoacoustic basis for the contrast. Buder, Chorna, Oller, and Robinson (2008)6 used the labels of "loft," "modal," and "pulse" for distinct vibratory regimes that appear to be identifiable based on spectrographic inspection of harmonic structure and auditory judgments in infants, but this work did not supply acoustic measurements to verify which of these nominally labeled regimes resembled adult registers. In this report, we identify clear transitions between registers within infant vocalizations and measure these registers and their transitions for fo and relative harmonic amplitudes (H1-H2). By selectively sampling first-year vocalizations, this manuscript quantifies acoustic patterns that correspond to vocal fold vibration types not previously cataloged in infancy. Results support a developmental basis for vocal registers, revealing that a well-developed ligament is not needed for loft-modal quality shifts as seen in harmonic amplitude measures. Results also reveal that a distinctively pulsatile register can occur in infants at a much higher fo than expected on psychoacoustic grounds. Overall results are consistent with cover tissues in infancy that are, for vibratory purposes, highly compliant and readily detached.

The anatomical evolution of the thyroid cartilage from childhood to adulthood: A computed tomography evaluation.

OBJECTIVE: To enhance knowledge and understanding of the laryngeal framework maturation in different age groups and genders. STUDY DESIGN: Cohort imaging study. SETTING: Tertiary academic referral center. METHODS: Computed tomography neck scans of 283 patients aged 8 to 20 years were studied. The interlaminae angle (ILA) of the thyroid cartilage at the level of the vocal folds, the anterior projection (angulation) of the thyroid cartilage (TC), and the degree of calcifications were evaluated and compared in sequential age groups of both genders. RESULTS: Neck scans of 171 males and 112 females were reviewed. The average ILA was 76.45° ± 14.2 and 94.25° ± 10.2 for males and females, respectively (P < 10-25). In the female group, the mean angle was relatively constant (91-970) in all age groups, whereas in the male groups the angle decreased with age (920-670) (r = -0.9, P < 0.005) The most significant decrease was measured in the 14- to 15-year age group. The thyroid prominence was significantly more anteriorly angulated in males. The angle in the female age groups was constant (170.1°), and the angle in males decreased with age (161.47°) (P = 0.000008). Calcifications were more prominent at the posterior portion of the cartilage in both genders and increased with age. CONCLUSION: Structural diversities of the TC begin in adolescent males because the thyroid cartilage grows anteriorly with a narrower ILA and with a greater anterior angulation. Our study shows that these changes, along with the degree of laryngeal cartilages calcification in both genders, occur as a continuum throughout puberty. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:E354-E358, 2017.

Fundamental Frequency of Crying in Two-month-old Boys and Girls: Do Sex Hormones During Mini-puberty Mediate Differences?

OBJECTIVE: To evaluate whether the puberty-like sex hormone surge during the first months of life (mini-puberty) affects fundamental frequency (fo) in infant crying as one would derive from hormone influences on voice in adults. STUDY DESIGN: Populational prospective study. PARTICIPANTS: Twenty healthy normal-hearing infants (nine boys) were recruited for participation. METHODS: Spontaneously uttered cries were collected from each infant at 8 weeks of age. The cries were acoustically analyzed for mean fo and fo range. The fo properties were correlated to the average serum levels of bioavailable estradiol (E2) (mean E2/sex hormone-binding globulin [SHBG]) and testosterone (T) (mean T/SHBG) across the second month of life. RESULTS: Whereas no significant hormone effect was found for mean fo, a significant negative correlation (r = -0.55) was found between fo range and mean E2/SHBG. No indication for a T influence on fo features was found at this age. Although girls showed a slightly higher mean E2 concentration than boys did, the observed differences in cry fo range were judged to be reflective of an infant's serum concentration of E2 rather than a sex-based difference. CONCLUSION: In the absence of laryngeal size differences between female and male infants, the result was interpreted as indicative of an E2 influence on viscoelastic properties of the vocal folds. In our opinion, the investigation of young infants' vocalizations during the early postnatal surge of sex steroids (mini-puberty) may advance our understanding of the mechanisms mediating average sex differences in vocal development and early communication.

Using attenuation coefficients from optical coherence tomography as markers of vocal fold maturation.

OBJECTIVES/HYPOTHESIS: Optical coherence tomography (OCT) is a promising technology to noninvasively assess vocal fold microanatomy. The goal of this study was to develop a methodology using OCT to identify quantifiable markers of vocal fold development. STUDY DESIGN: In vivo study. METHODS: A two-step process was developed to reproducibly image the midmembranous vocal fold edge of 10 patients younger than 2 years and 10 patients between 11 and 16 years of age using OCT. An image analysis algorithm was implemented to extract OCT-derived A-lines for each patient. These A-lines were divided into three zones according to apparent changes in slope. Relative attenuation coefficients, or tissue- and system-dependent parameters that describe the rate at which optical signal decays, were calculated for each zone. RESULTS: Young patients had distinct relative attenuation coefficients in zone 1 (P < .0001), whereas zones 2 and 3 were indistinct (P = .1129). Older patients had distinct relative attenuation coefficients in zones 1, 2, and 3 (P < .0370). Between age groups, relative attenuation coefficients were different in zones 2 and 3 (P < .0001, P = .0315, respectively) and indistinct in zone 1 (P = .1438). CONCLUSIONS: Relative attenuation coefficients can be used as markers of vocal fold development. Differences in relative attenuation coefficients likely represent changes in extracellular matrix structure within the lamina propria and may become useful for guiding treatment of voice disorders in the pediatric population. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E218-E223, 2016.

LANGUAGE DEVELOPMENT. The developmental dynamics of marmoset monkey vocal production.

Human vocal development occurs through two parallel interactive processes that transform infant cries into more mature vocalizations, such as cooing sounds and babbling. First, natural categories of sounds change as the vocal apparatus matures. Second, parental vocal feedback sensitizes infants to certain features of those sounds, and the sounds are modified accordingly. Paradoxically, our closest living ancestors, nonhuman primates, are thought to undergo few or no production-related acoustic changes during development, and any such changes are thought to be impervious to social feedback. Using early and dense sampling, quantitative tracking of acoustic changes, and biomechanical modeling, we showed that vocalizations in infant marmoset monkeys undergo dramatic changes that cannot be solely attributed to simple consequences of growth. Using parental interaction experiments, we found that contingent parental feedback influences the rate of vocal development. These findings overturn decades-old ideas about primate vocalizations and show that marmoset monkeys are a compelling model system for early vocal development in humans.

Evaluation of true vocal fold growth as a function of age.

OBJECTIVE: To evaluate change in true vocal fold length as a function of age. STUDY DESIGN: Prospective study. SETTING: Tertiary aerodigestive center. SUBJECTS AND METHODS: In total, 205 patients (aged 1 month to 20 years), of whom 87 (42.4%) were female and 118 (57.6%) male, were included. Lengths of the total vocal fold (TVFL), membranous vocal fold (MVFL), and cartilaginous vocal fold (CVFL) were measured during direct laryngoscopy. Membranous-to-cartilaginous (M/C) ratios were calculated. RESULTS: For patients younger than 1 year, mean (SD) MVFL was 4.4 (1.3) mm for females and 4.9 (1.8) mm for males. At age 17 years, mean (SD) MVFL was 12.3 (2.1) mm for females and 14.0 (1.4) mm for males. Mean TVFL, MVFL, and CVFL increased an average of 0.7 mm, 0.5 mm, and 0.2 mm per year in linear fashion, respectively (linear regression, P < .0001). The M/C ratio did not significantly change with age (P = .33). Mean TVFL, MVFL, and CVFL showed no statistical difference between males and females (P = .27, .11, and .75, respectively). CONCLUSION: This is the largest longitudinal pediatric study specifically examining vocal fold length as a function of age. Each length of the true vocal fold appeared to linearly increase for both females and males. The M/C ratio remained relatively constant, unlike previously reported data, possibly due to in vivo vs cadaveric measurements. These findings suggest that critical periods of development in females and males are not explainable by changes in vocal fold length alone, and other factors such as vocal fold layers need further exploration.

Nasotracheal intubation depth in paediatric patients.

BACKGROUND: The aim of this study was to compare intubation depth using the Microcuff paediatric endotracheal tube (PET) placed with the intubation depth mark between the vocal cords with that of different published formulae/recommendations for nasotracheal intubation depth in children. METHODS: Children aged from birth to 10 yr undergoing elective surgery with nasotracheal intubation were included. Tracheal tubes were adjusted according to the intubation depth mark between the vocal cords using direct laryngoscopy. Nasal intubation depth was recorded and the distance 'tube tip to carina' was measured endoscopically. Based on the recorded nasal intubation depth and measured distance 'tube tip to carina', the position of tube tip and cuff was calculated according to six published formulae/recommendations. RESULTS: Seventy-six children were studied. For the Microcuff PET, the median tube tip advancement within the trachea was 52.9% (41.1-73.8%) of tracheal length. The shortest distance from the 'tube tip to carina' was 15 mm for a 3.5 mm internal diameter tube. If the six published formulae/recommendations had been used, this would have resulted in endobronchial tube placement in up to 9.1% of cases, and the tube tip would have been placed above the glottis in up to 2.6% of cases. The upper border of the cuff would have been placed in the subglottic area in up to 42.1% of cases and in a supraglottic position in up to 63.2% of cases. CONCLUSIONS: This study indicates that nasal intubation with the intubation depth mark placed between the vocal cords was superior to formula-based nasotracheal tube positioning. The latter would result in a high rate of endobronchial intubations, excessively high cuff positions and even tracheal extubations.

Measurement of glottal cycle characteristics between children and adults: physiological variations.

OBJECTIVES: The aim of this study was to quantify phases of the vibratory cycle using measurements of glottal cycle quotients and glottal cycle derivatives, in typically developing prepubertal children and young adults with the use of high-speed digital imaging (HSDI). METHODS: Vocal fold vibrations were recorded from 27 children (age range 5-9 years) and 35 adults (age range 21-45 years), with HSDI at 4000 frames per second for sustained phonation. Glottal area waveform measures of Open Quotient, Closing Quotient, Speed Index (SI), Rate Quotient, and Asymmetry Quotient (AsyQ) were computed. Glottal cycle derivatives of Amplitude Quotient (AQ) and Maximum Area Declination Rate (MADR) were also computed. Group differences (adult females, adult males, and children) were statistically investigated for mean and standard deviation values of the glottal cycle quotients and glottal cycle derivatives. RESULTS: Children exhibited higher values of SI and AsyQ and lower values of MADR compared with adult males. Children exhibited the highest mean value and lowest variability in AQ compared with adult males and females. Adult males showed lower values of SI, AsyQ, AQ, and higher values of MADR compared with adult females. CONCLUSIONS: Glottal cycle vibratory motion in children is functionally different compared with adult males and females, suggesting the need for development of children specific norms for both normal and disordered voice qualities.

Vocal fold fibroblast response to growth factor treatment is age dependent: results from an in vitro study.

INTRODUCTION: Vocal fold (VF) fibroblasts (VFFs) are the central target in developing new strategies for treatment of VF injury and scarring. Nevertheless, only little is known about the basic biological characteristics of these cells. The aim of this study was to explore the impact of age of VFFs on the response to external growth factor stimulation. STUDY DESIGN: In vitro cell study using a rat model. METHODS: VFFs were extracted from young and aged rat VF 3 months after establishing unilateral injury. Resulting scar fibroblasts (SFs) and normal fibroblasts (NFs) were subsequently cultured separately with or without the addition of hepatocyte growth factor (HGF). After 24 and 72 hours, the production of hyaluronic acid (HA) was examined in the supernatant culture media using enzyme-linked immunosorbent assay. RESULTS: Only cultured SF and NF from young animals could be stimulated significantly in the production of HA by HGF. Within these, average percentage increase was higher in NF compared with SF. CONCLUSION: The response of VFFs in cell culture to growth factors stimulation is highly depending on the age of the animals. This is another step in a nearer characterization of scar VFF and could furthermore be an important point when estimating the success of an intervention. Age-depending effects must be considered as an important factor in developing possible therapeutic agents for VF scarring.

Quantitative distinction of unique vocal fold subepithelial architectures using optical coherence tomography.

OBJECTIVES: The primary objective of this study was to quantitatively analyze ex vivo porcine, fetal human, and adult human vocal folds by use of optical coherence tomography (OCT). A secondary objective was to quantitatively discriminate among 1-, 2-, and 3-layer lamina propria structures. METHODS: We performed an analysis of the vocal folds of 10 adult pig, 3 adult human, and 2 fetal human vocal fold specimens using OCT and histologic techniques. We present a quantitative comparison of the OCT results and histologic findings. RESULTS: We found that OCT allowed for the visualization of the subepithelial vocal fold architecture of all imaged tissue, and that it revealed distinct characteristic signal intensities for each type of specimen. CONCLUSIONS: Optical coherence tomography was developed for in vivo imaging of biological microstructures. This study demonstrates the ability of OCT to differentiate between the vocal fold architectures of 3 histologically distinct types of vocal folds. Future studies aim to develop a quantitative optical imaging algorithm that can be used to facilitate an in vivo longitudinal clinical investigation of the changes that occur in this layered structure over time and maturation.

Voice training and changing weight--are they reflected in speaking fundamental frequency, voice range, and pitch breaks of 13-year-old girls? A longitudinal study.

OBJECTIVE: Assessment of the voice-change progress of 20 girls (12-13 years) over 1 year by observing changes in speaking fundamental frequency (SFo), voice range, and register pitch breaks in the context of weight, height, voice training, and self-perception. STUDY DESIGN: One-year longitudinal collective case study. METHOD: Twenty girls were recorded at the beginning and end of a year; nine girls were recorded another three times. SFo, vocal range, and characteristics were analyzed and interactions between these data assessed against weight and height to indicate pubertal development, and to test the hypothesis that changes in weight, height, SFo, and pitch breaks were related. Effects of training and the girls' self-perception of their voice use were also assessed. RESULTS: Vocal characteristics changed as the girls passed through different weight ranges. During 47.5-52.4 kg (called band 2) and 52.4-57.5 kg (band 3), there was progressive contraction of vocal range and in some girls a slight rise in SFo between recording times 1 and 5. Both high- and low-pitch breaks were present in 45% of girls' voices. Girls in band 4 (<57.5 kg) had an increased vocal range, and pitch breaks in vocal-range areas that indicated the development of adult vocal registers. In this study, voice-trained girls were heavier, had higher SFo, used wider speech-range inflection, had a higher vocal range, and greater voice-use confidence; all girls lost confidence in their voice use over the year. CONCLUSIONS: In this longitudinal study of twenty 13-year-old girls, voice changes in SFo, vocal range, and pitch-break frequency were synchronous with certain weight ranges. Girls with training registered higher maximum phonational frequency and were more confident in their voice use than girls without training.

Vocal tract dimensional development of adolescents: an acoustic reflection study.

OBJECTIVE: The purpose of the study was to investigate the effects of age and gender on adolescents' vocal tract dimensional development with acoustic reflection technology (ART). METHODS: A total of ninety-five male and female adolescents aged between 10 and 18 divided into three age groups were tested with acoustic reflection technology (ART) and acoustic program to secure their vocal tract dimensional parameters and the vowel formant frequencies. RESULTS: Significant age and gender effects were found not only in vocal tract length, but also segmental volumetric measurements, as well as the vowel formant frequencies. CONCLUSIONS: The findings of this study have provided insights on the developmental trend of adolescents' vocal tracts. The study has also offered a preliminary anatomical database of adolescents' vocal tract dimensional growth for otolaryngologists, clinical anatomists, speech therapists and other health professionals of swallowing, respiration and communicative disorders.

Functional histology of the macula flava in the human vocal fold--Part 2: Its role in the growth and development of the vocal fold.

OBJECTIVE: This study aims to clarify the role of the maculae flavae (MFe) during growth and development of the human vocal fold mucosa (VFM). METHODS: Our current results concerning the MFe in the human newborn, infant, and child VFM are summarized. RESULTS: Newborns already had immature MFe at the same sites as adults. They were composed of dense masses of vocal fold stellate cells (VFSCs), whereas extracellular matrix components were sparse. VFSCs in the newborn MFe had already started synthesizing extracellular matrices (EM). During infancy, the EM synthesized in the MFe appeared in the VFM to initiate the formation of the three-dimensional extracellular matrix structure of the human VFM. During childhood, MFe including VFSCs continued to synthesize EM such as collagenous, reticular, and elastic fibers, and hyaluronic acid (glycosaminoglycan), which are essential for the human VFM as a vibrating tissue. The MFe in newborns, infants and children were related to the growth and development of the human VFM. CONCLUSION: Human MFe including VFSCs were inferred to be involved in the metabolism of EM, essential for the viscoelasticity of the human VFM, and are considered to be an important structure in the growth and development of the human VFM.

Electrolaryngographically revealed aspects of the voice source in singing.

This paper provides an overview of some of the ways in which the output from the electrolaryngograph enables aspects of the vibrating vocal folds during singing and speech to be monitored non-invasively. This paper was presented at the British Voice Association Symposium in honour of the work of Adrian Fourcin on 9 May 2009 at University College London; Adrian developed the electrolaryngograph itself and researched and promoted its use as a practical device for use both clinically and in the speech sciences laboratory. Here, the application of the electrolaryngograph for voice source investigations relating to singing in a number of situations is considered, including: singing in different styles, tuning accuracy and vocal blend in unaccompanied (a capella) quartet singing, and longitudinal aspects of voice development in girl choristers.