Kate Lesciotto

Dr Yann Heuzé

Dr Ethylin Wang Jabs

Dr Joseph M Bernstein

Dr Joan T Richtsmeier

Ms Kate

MS M Lesciotto

Choanal Atresia and Craniosynostosis: Development and Disease

published or not. The documents may come

Introduction

We present a review of case reports that link craniosynostosis and choanal atresia to highlight the uncertainty of a choanal atresia diagnosis in pediatric craniosynostosis patients and provide anatomical data from human and mouse to more fully define choanal and associated dysmorphologies. The lack of a precise definition of choanal atresia in the current craniosynostosis literature results in an unclear set of standards for the diagnosis of choanal dysmorphologies. The developmental genetic significance of the association of choanal atresia and craniosynostosis and the implications for developing appropriate therapeutics requires a clear understanding of these anomalies.

The Human Choanae

In humans, the choanae are defined in several ways. Osteologically, the choanae are the posterior openings of the right and left nasal passages that are bordered medially by the posterior border of the vomer, superiorly by the sphenoid body, laterally by the medial pterygoid plates, and inferiorly by the horizontal plate of the palatine bones [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF] (Fig. 1). An anatomical definition includes these osteological borders of the choanae, or posterior nares, while incorporating the surrounding soft tissues: the choanae are the pair of posterior apertures of the nasal cavity that open into the nasopharynx. Each choana can be defined functionally, as an internal nostril, connecting the nasal air space and the posterior roof of the pharyngeal cavity (Fig. 2). Study of extant jawed fishes and fossil vertebrates show that choanae evolved from a condition in which anterior and posterior external nostrils functioned without a connection between the nasal sac and the oral cavity [START_REF] Zhu | The origin of the internal nostril in tetrapods[END_REF] . The tetrapod choanae ("internal nostrils") are homologous to the posterior external nostrils of jawed fishes [START_REF] Zhu | The origin of the internal nostril in tetrapods[END_REF] and are a key feature of the evolution of tetrapods, a group that includes, reptiles, mammals, and humans. The tetrapod respiratory system appeared with the evolution of the palate separating the nasal and oral respiratory systems. Only tetrapods possess choanae [START_REF] Zhu | The origin of the internal nostril in tetrapods[END_REF] . Embryogenesis of the choanae is complex, characterized by several distinct developmental periods, each requiring the precise spatiotemporal coordination of the development of diverse tissues and functioning spaces before the final structure and function are reached (Fig. 3). At the end of the seventh week of prenatal ontogeny, the medial nasal prominences fuse [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF] , providing the foundation for the primary palate [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF][START_REF] Sperber | Craniofacial Embryogenetics and Development[END_REF] . The posterior portion of the intermaxillary process becomes the oro-olfactory, oronasal, or nasobuccal membrane, which separates the developing olfactory sac from the oral cavity [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF][START_REF] Hengerer | Choanal atresia: Embryologic analysis and evolution of treatment, a 30-year experience[END_REF] . When this membrane ruptures, the primary choanae are formed, permitting communication between the nasal and oral cavities [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF][START_REF] Kim | Early development of the nose in human embryos: a stereomicroscopic and histologic analysis[END_REF] . At this stage, the lateral palatal shelves are still oriented vertically [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF][START_REF] Kim | Early development of the nose in human embryos: a stereomicroscopic and histologic analysis[END_REF] . As these shelves transition downward to their final horizontal position, the remnants of the primary choanae become the incisive foramen, the primary palate fuses to the secondary palate posteriorly, the right and left lateral shelves of the secondary palate fuse along the midline, and the posterior or secondary choanae are formed and shifted posteriorly following this progressive fusion [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF][START_REF] Hengerer | Choanal atresia: Embryologic analysis and evolution of treatment, a 30-year experience[END_REF][START_REF] Kim | Early development of the nose in human embryos: a stereomicroscopic and histologic analysis[END_REF][START_REF] Tamarin | The formation of the primitive choanae and the junction of the primary and secondary palates in the mouse[END_REF][START_REF] Mankarious | Craniofacial and upper airway development[END_REF] . During this time, the nasal septum has formed from the roof of the nasal cavity to meet the superior surfaces of the primary and secondary palates along the midline, dividing the left and right nasal cavities [START_REF] Jankowski | The Evo-Devo Origin of the Nose, Anterior Skull Base and Midface[END_REF] . The completion of this process results in separation of the right and left nostrils and separation of the nasal and oral cavities, with the secondary choanae defining the posterior aspect of the left and right nasal cavities immediately rostral to the nasopharynx. For the purposes of this article, the secondary choanae are referred to generally as the choanae.

Choanal Atresia -Definition, development and diagnosis

Errors in timing, organization, or development of the palate can give rise to numerous dysmorphic conditions, including various degrees of clefting of the hard and/or soft palate [START_REF] Sperber | Craniofacial Embryogenetics and Development[END_REF] . Choanal atresia is a less common, though medically significant, anomaly associated with errors of development of the nasal cavity and palate. Choanal atresia is defined as the complete obstruction of the posterior nasal apertures (choanae) by osseous tissue, either alone or in combination with non-osseous tissue [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Harney | Pediatric Surgery[END_REF] . This blockage may occur unilaterally or bilaterally and results in a lack of communication of the nasal cavity with the pharyngeal cavity via the nasopharynx 1 , thereby preventing inhalation and exhalation of air through the affected nasal passage(s). Two major osteological deformities have been described in choanal atresia: 1) a medialization of the medial pterygoid plates; and 2) a thickening of the posterior vomer [START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Aslan | Comparison of nasal region dimensions in bilateral choanal atresia patients and normal controls: A computed tomographic analysis with clinical implications[END_REF][START_REF] Cheung | Comparison of craniofacial skeletal characteristics of infants with bilateral choanal atresia and an age-matched normative population: computed tomography analysis[END_REF] . Either of these deformations can lead to a narrowing of the choanae, potentially resulting in complete obstruction of the choanae. Several developmental theories are commonly cited in the formation of choanal atresia: (1) persistence of the buccopharyngeal membrane from the foregut; (2) persistence or abnormal location of mesoderm forming adhesions in the nasochoanal region; (3) persistence of the nasobuccal membrane of Hochstetter; and (4) misdirection of neural crest cell migration and subsequent flow of mesoderm [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Hengerer | Choanal atresia: Embryologic analysis and evolution of treatment, a 30-year experience[END_REF][START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Harney | Pediatric Surgery[END_REF][START_REF] Bergstrom | Posterior choanal atresia -a syndromal disorder[END_REF] . However, none of these provides a precise explanation for obstruction or minimization of the size of the choanal openings by developmental processes, and to date, there has been no definitive evidence supporting one theory over the others.

Significantly, choanal atresia must be differentiated from choanal stenosis, a diagnosis defined as the narrowing of the posterior choanae without complete obstruction [START_REF] Lowe | Midface anomalies in children[END_REF] , and from nasal pyriform aperture stenosis, which involves narrowing of the skeletal borders of the anterior nasal cavity [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Brown | Congenital nasal pyriform aperture stenosis[END_REF][START_REF] Keller | Choanal atresia, CHARGE association, and congenital nasal stenosis[END_REF] . Precise definitions are required to correct common errors that incorporate narrowing or incomplete obstruction of the choanae within the definition of choanal atresia or that conflate choanal atresia with choanal stenosis (see, e.g., [START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Ramsden | Choanal Atresia and Choanal Stenosis[END_REF][START_REF] Meyers | Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans[END_REF][START_REF] Wilkes | A recurrent mutation, ala391glu, in the transmembrane region of FGFR3 causes Crouzon syndrome and acanthosis nigricans[END_REF] ). The potential for the misdiagnosis of choanal atresia has been recognized in pediatric patients with major craniofacial anomalies since these conditions routinely include some form of midfacial retrusion. Airway obstruction is common in craniofacial syndromes due to potential maldevelopment of the palate (floor of the pyriform aperture), the nasal airway, the nasopharynx, or the entire midfacial skeleton in the production of midfacial dysmorphogenesis [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Sculerati | Airway management in children with major craniofacial anomalies[END_REF][START_REF] Buchman | Syndromic craniosynostosis[END_REF][START_REF] Allanson | Elements of morphology: Standard terminology for the head and face[END_REF] .

Choanal atresia is typically suspected in infants exhibiting respiratory distress, particularly when feeding [START_REF] Aslan | Comparison of nasal region dimensions in bilateral choanal atresia patients and normal controls: A computed tomographic analysis with clinical implications[END_REF][START_REF] Cheung | Comparison of craniofacial skeletal characteristics of infants with bilateral choanal atresia and an age-matched normative population: computed tomography analysis[END_REF] . Bilateral choanal atresia in neonates presents an emergent situation, as infants are obligate nasal breathers. Bilateral choanal atresia leads to cyclic cyanosis relieved by crying which facilitates mouth breathing [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Harney | Pediatric Surgery[END_REF] . While truly complete obstruction of the posterior choanae can only be confirmed through diagnostic imaging or endoscopy, choanal atresia is often diagnosed by the inability to cannulate the nasal passage with a small catheter, a procedure that cannot definitively distinguish partial stenosis or complete obstruction of the choanae [START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Aslan | Comparison of nasal region dimensions in bilateral choanal atresia patients and normal controls: A computed tomographic analysis with clinical implications[END_REF][START_REF] Keller | Choanal atresia, CHARGE association, and congenital nasal stenosis[END_REF][START_REF] Buchman | Syndromic craniosynostosis[END_REF][START_REF] Neskey | Nasal, Septal, and Turbinate Anatomy and Embryology[END_REF] . The incidence of choanal atresia ranges from 1 in 5000-8000 live births, with a 2:1 higher occurrence in females [START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Cheung | Comparison of craniofacial skeletal characteristics of infants with bilateral choanal atresia and an age-matched normative population: computed tomography analysis[END_REF][START_REF] Neskey | Nasal, Septal, and Turbinate Anatomy and Embryology[END_REF][START_REF] Burrow | Characterization of congenital anomalies in individuals with choanal atresia[END_REF] . Unilateral choanal atresia is slightly more common than bilateral atresia, while bilateral atresia is more common when other craniofacial malformations are present [START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Cheung | Comparison of craniofacial skeletal characteristics of infants with bilateral choanal atresia and an age-matched normative population: computed tomography analysis[END_REF][START_REF] Neskey | Nasal, Septal, and Turbinate Anatomy and Embryology[END_REF] .

In an early review, Durward and colleagues (1945) defined choanal atresia as a very rare condition and concluded that the association between choanal atresia and other syndromic craniofacial dysmorphologies was no more than spurious [START_REF] Durward | Congenital choanal atresia[END_REF] . Improvements in diagnostic imaging and neonatal care have permitted researchers to make the explicit link between choanal atresia and a number of craniofacial disorders, most notably CHARGE syndrome, with an estimated 7-29% of choanal atresia patients also being diagnosed with CHARGE [START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF] . Syndromic craniosynostosis patients make up another core subset of patients diagnosed with choanal atresia, with specific associations made between choanal atresia and Antley-Bixler, Apert, Beare-Stevenson, Crouzon, Crouzonodermoskeletal (Crouzon with acanthosis nigricans), Jackson-Weiss, and Pfeiffer syndromes [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Lowe | Midface anomalies in children[END_REF][START_REF] Keller | Choanal atresia, CHARGE association, and congenital nasal stenosis[END_REF][START_REF] Ramsden | Choanal Atresia and Choanal Stenosis[END_REF][START_REF] Cohen | Craniosynostosis: Diagnosis, Evaluation, and Management[END_REF][START_REF] Arnaud-López | Crouzon with acanthosis nigricans. Further delineation of the syndrome[END_REF][START_REF] Hehr | Craniosynostosis syndromes: from genes to premature fusion of skull bones[END_REF] .

Choanal Atresia and Syndromic Craniosynostosis in Pediatric Patients

Craniosynostosis is a condition of complex etiology that always involves the premature fusion of one or multiple cranial sutures and includes various anomalies of the soft and hard tissues of the head [START_REF] Flaherty | Understanding craniosynostosis as a growth disorder[END_REF] . In cases of syndromic craniosynostosis, the closed suture occurs as part of a suite of symptoms or features, and mutations in a number of genes have been identified as being associated with these syndromes (see, e.g., [START_REF] Flaherty | Understanding craniosynostosis as a growth disorder[END_REF][START_REF] Heuzé | Closing the gap: genetic and genomic continuum from syndromic to nonsyndromic craniosynostoses[END_REF][START_REF] Lattanzi | Genetic advances in craniosynostosis[END_REF] ). The nearly 200 identified craniosynostosis syndromes account for approximately 15% of all craniosynostosis cases [START_REF] Flaherty | Understanding craniosynostosis as a growth disorder[END_REF] . Recent work stresses the complexity of craniosynostosis phenotypes even in cases of nonsyndromic (isolated) craniosynostosis, emphasizing that craniosynostosis conditions need to be defined not simply by premature suture closure, but more broadly as growth disorders that affect many different cell and tissue lineages [START_REF] Flaherty | Understanding craniosynostosis as a growth disorder[END_REF][START_REF] Heuzé | Closing the gap: genetic and genomic continuum from syndromic to nonsyndromic craniosynostoses[END_REF][START_REF] Heuzé | New insights into the relationship between suture closure and craniofacial dysmorphology in sagittal nonsyndromic craniosynostosis: Skull shape in isolated sagittal craniosynostosis[END_REF][START_REF] Heuzé | Unilateral and bilateral expression of a quantitative trait: asymmetry and symmetry in coronal craniosynostosis[END_REF][START_REF] Martínez-Abadías | Tissue-specific responses to aberrant FGF signaling in complex head phenotypes[END_REF] . Consequent to the broad developmental impact of the genes on which craniosynostosis-causing mutations are located (e.g., fibroblast growth factor receptors, TWIST), many craniofacial tissues are affected in craniosynostosis syndromes, including skeletal (bone and cartilage), muscular, neural, and circulatory structures. Facial dysmorphologies potentially associated with craniosynostosis syndromes include maxillary dysmorphogenesis resulting in a reduced midface, hypertelorism, exopthalmos, depressed or low nasal bridge, mandibular prognathism, cleft palate, and highly arched and/or constricted palate [START_REF] Cohen | Craniosynostosis: Diagnosis, Evaluation, and Management[END_REF][START_REF] Cohen Mm | A clinical study of the craniofacial features in Apert syndrome[END_REF][START_REF] Cunningham | Syndromic craniosynostosis: from history to hydrogen bonds[END_REF][START_REF] Johnson | Craniosynostosis[END_REF][START_REF] Nah | Phenotype profile of a genetic mouse model for Muenke syndrome[END_REF][START_REF] Heuzé | Quantification of facial skeletal shape variation in fibroblast growth factor receptor-related craniosynostosis syndromes: Facial shape in Fgfr-Related craniosynostoses[END_REF] . Any one of these structural anomalies has the potential to contribute to altering the position, size, shape, or patency of the choanae.

Craniosynostosis has been explicitly linked with choanal atresia in one of the seminal texts on the diagnosis and evaluation of craniosynostosis, noting that atresia or stenosis is an "expected" clinical finding in craniosynostosis syndromes, particularly where there have been structural rearrangements in the cranial base [START_REF] Cohen | Craniosynostosis: Diagnosis, Evaluation, and Management[END_REF] , a region of the skull that forms endochondrally from a complex series of cartilages that underlie the brain. Additional links between syndromic craniosynostosis and choanal atresia can be found in review and research articles throughout the clinical literature (see, e.g., [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Lowe | Midface anomalies in children[END_REF][START_REF] Keller | Choanal atresia, CHARGE association, and congenital nasal stenosis[END_REF][START_REF] Ramsden | Choanal Atresia and Choanal Stenosis[END_REF][START_REF] Hehr | Craniosynostosis syndromes: from genes to premature fusion of skull bones[END_REF][START_REF] Cunningham | Syndromic craniosynostosis: from history to hydrogen bonds[END_REF] Of the 54 case reports reviewed, none included a definition of choanal atresia, and several provide descriptions suggesting that the condition may have more likely been choanal stenosis [START_REF] Meyers | Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans[END_REF][START_REF] Wilkes | A recurrent mutation, ala391glu, in the transmembrane region of FGFR3 causes Crouzon syndrome and acanthosis nigricans[END_REF][START_REF] Erten | Respiratory and cranial complications during anaesthesia in Pfeiffer syndrome[END_REF] . For example, various authors reported (emphases added):

• "all four of our patients exhibited choanal atresia (narrowed nasal passage)" 19

• "incomplete choanal atresia led to respiratory difficulties" 20

• condition was first labeled "choanal atresia" and later as "choanal hypoplasia" [START_REF] Erten | Respiratory and cranial complications during anaesthesia in Pfeiffer syndrome[END_REF] , the former being a diagnostic category and the latter being a description that suggests the developmental basis of this anomaly.

Additionally, the methods of evaluation and diagnosis were often not indicated, and the fundamental differences among diagnostic tools were not discussed by these authors. Only eight cases reported the use of CT imaging to confirm the choanal atresia diagnosis [START_REF] Barge-Schaapveld | Beare-Stevenson syndrome: Two Dutch patients with cerebral abnormalities[END_REF][START_REF] Scheid | Tracheal cartilaginous sleeve in Crouzon syndrome[END_REF][START_REF] Wenger | Beare-Stevenson syndrome: Two new patients, including a novel finding of tracheal cartilaginous sleeve[END_REF][START_REF] Sharda | A newborn with acanthosis nigricans: Can it be Crouzon syndrome with acanthosis nigricans?[END_REF][START_REF] Hassell | Antley-Bixler syndrome: report of a patient and review of literature[END_REF][START_REF] Hosalkar | The Antley-Bixler syndrome: two new cases[END_REF][START_REF] Ron | A case of Beare-Stevenson syndrome with unusual manifestations[END_REF][START_REF] Dunmade | Intranasal endoscopic repair of bilateral choanal atresia in a male newborn with Crouzon's syndrome[END_REF] , while others cited Doppler evidence 50 , choanagraphy [START_REF] Pius | Apert syndrome: A case report and review of literature[END_REF] , inability to pass a naso-gastric tube through the posterior choanae [START_REF] Shrestha | Apert syndrome (acrocephalosyndactyly): A rare syndromic craniosynostosis[END_REF] , simple reference to "imaging" [START_REF] Hosalkar | The Antley-Bixler syndrome: two new cases[END_REF] , and pharyngiogram [START_REF] Alpers | Hemangiomatous anomaly of bone in Crouzon's syndrome -case report[END_REF] .

Although CT imaging was mentioned in seven additional case reports, those reports did not include an indication of whether the scan was utilized in the choanal atresia diagnosis [START_REF] Alpers | Hemangiomatous anomaly of bone in Crouzon's syndrome -case report[END_REF][START_REF] Hall | Beare-Stevenson cutis gyrata syndrome[END_REF][START_REF] Hsu | Prenatal sonographic appearance of Beare-Stevenson cutis gyrata syndrome: two-and three-dimensional ultrasonographic findings[END_REF][START_REF] Ito | A cloverleaf skull syndrome probably of Beare-Stevenson type associated with Chiari malformation[END_REF][START_REF] Kroczek | Cloverleaf skull associated with Pfeiffer syndrome: pathology and management[END_REF][START_REF] Mathijssen | Pfeiffer's syndrome resulting from an S351C mutation in the fibroblast growth factor receptor-2 gene[END_REF][START_REF] Oyamada | Pfeiffer syndrome type 2: case report[END_REF] .

Another nine reports mentioned various types of surgical intervention in which direct visualization may have been possible, but no explicit description of the surgical evaluation was given [START_REF] Hall | Beare-Stevenson cutis gyrata syndrome[END_REF][START_REF] Kroczek | Cloverleaf skull associated with Pfeiffer syndrome: pathology and management[END_REF][START_REF] Lo | Airway obstruction in severe syndromic craniosynostosis[END_REF][START_REF] Adolphs | Antley-Bixler-Syndrome -Staged management of craniofacial malformations from birth to adolescence -A case report[END_REF][START_REF] Vargas | Beare-Stevenson syndrome: Two South American patients with FGFR2 analysis[END_REF][START_REF] Oldridge | De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome[END_REF][START_REF] Boynukalın | Prenatal diagnosis of Apert syndrome: a case report[END_REF][START_REF] Schweitzer | Subtle radiographic findings of achondroplasia in patients with Crouzon syndrome with acanthosis nigricans due to an Ala391Glu substitution in FGFR3[END_REF][START_REF] Suslak | Crouzon syndrome with periapical cemental dysplasia and acanthosis nigricans: the pleiotropic effect of a single gene? Birth Defects[END_REF] . These reports also varied widely in the detail of the description of cooccurring facial anomalies that might contribute to respiratory difficulties. It is important to note that, unless the above-referenced case reports included images of the diagnostic scans, it is impossible to say for certain whether the suggested choanal atresia was correctly diagnosed.

Looking Forward

The clear implication of the case reports (Table 1) is the need for a consistent application of an invariant clinical definition of choanal atresia that is distinct from choanal stenosis. The term "choanal atresia" was used in a number of these case reports, yet the condition described may actually be choanal stenosis. Without review of each described patient's medical records and associated diagnostic images and results, we are only able to note that the diagnosis is not well supported based on the published information and cannot definitively state whether any of these choanal atresia diagnoses are truly erroneous. Other reports simply group the conditions together and report a finding of "choanal stenosis/ atresia." Although options may be similar from a treatment perspective, understanding choanal stenosis and atresia as potentially different pathologies with distinct etiologies requires more precise descriptions and further research. While several craniofacial textbooks and journal articles provide clear definitions of choanal atresia [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Kwong | Current Updates on Choanal Atresia[END_REF][START_REF] Corrales | Choanal atresia: current concepts and controversies[END_REF][START_REF] Harney | Pediatric Surgery[END_REF][START_REF] Brown | Congenital nasal pyriform aperture stenosis[END_REF] , many authors either omit a definition from published case reports or fail to explicitly match a given definition to their clinical observations and reports. Additionally, while medical CT is acknowledged to be the gold standard for the diagnosis of choanal atresia [START_REF] Adil | Congenital nasal obstruction: clinical and radiologic review[END_REF][START_REF] Harney | Pediatric Surgery[END_REF][START_REF] Lowe | Midface anomalies in children[END_REF][START_REF] Keller | Choanal atresia, CHARGE association, and congenital nasal stenosis[END_REF][START_REF] Ramsden | Choanal Atresia and Choanal Stenosis[END_REF][START_REF] Brown | The evaluation of choanal atresia by computed tomography[END_REF] , the vast majority of published case reports either fail to report the use of this preferred diagnostic methodology, or utilize less reliable techniques that may erroneously lead to a choanal atresia diagnosis when choanal stenosis or other choanal or nasal dysmorphology is present. Sculerati and colleagues' previous study of over 250 pediatric patients with major craniofacial anomalies produced results that support our observations, finding that choanal atresia was often misdiagnosed when respiratory difficulties were actually being caused by nasal obstructions secondary to midfacial retrusion [START_REF] Sculerati | Airway management in children with major craniofacial anomalies[END_REF] . In addition to the need for a better understanding of the facial dysmorphologies associated with midfacial retrusion (hypoplasia, flattening, dysgenesis), further research should be directed towards the investigation of the relationship between choanal stenosis and choanal atresia and whether they are distinct abnormalities or represent unique conditions along a continuum of choanal dysmorphogenesis. Given the state of the existing literature, it is recommended that case reports and research articles focusing on choanal atresia provide both an explicit definition of the condition, as well as details regarding the methodology used to detect and diagnose the condition. Recent caution regarding radiation exposure when using CT as a primary diagnostic tool [START_REF] Chen | Risk of malignancy associated with head and neck CT in children: a systematic review[END_REF] provides a timely opportunity to refine both the clinical definition of choanal atresia, as well as to develop a new standard for detection and diagnosis.

Research focused on choanal development, structure, and morphology in humans (especially within the pediatric craniosynostosis syndrome population) and animal models is needed to better understand the true incidence of choanal atresia within this patient population. Several studies have reported nasal airway volume or morphology in pediatric choanal atresia patients [START_REF] Aslan | Comparison of nasal region dimensions in bilateral choanal atresia patients and normal controls: A computed tomographic analysis with clinical implications[END_REF][START_REF] Cheung | Comparison of craniofacial skeletal characteristics of infants with bilateral choanal atresia and an age-matched normative population: computed tomography analysis[END_REF] , but little work has been done to quantify or describe choanal or nasal airway morphology in syndromic craniosynostosis patients. Perhaps more importantly, there have been few serious attempts to tie craniosynostosis conditions to choanal atresia developmentally or by molecular causation.

A recent analysis of 3D medical CT scans comprising children diagnosed with Apert, Pfeiffer, Muenke, or Crouzon syndrome and typically developing children (aged 0-23 months) without craniosynostosis who underwent CT imaging for unrelated conditions (e.g., seizures) provides information about differences in facial skeletal shape among craniosynostosis syndromes [START_REF] Heuzé | Quantification of facial skeletal shape variation in fibroblast growth factor receptor-related craniosynostosis syndromes: Facial shape in Fgfr-Related craniosynostoses[END_REF] . The 3D isosurfaces were reconstructed from the set of DICOM images [START_REF] Heuzé | Quantification of facial skeletal shape variation in fibroblast growth factor receptor-related craniosynostosis syndromes: Facial shape in Fgfr-Related craniosynostoses[END_REF] , and these 3DCTs were evaluated visually for the presence of choanal atresia. Of 33 individuals diagnosed with syndromic craniosynostosis, none had choanal atresia. Nasopharyngeal volume, including the ethmoidal air cells, was estimated for each patient using the segmentation editor of the software package Avizo 6.3 (Visualization Sciences Group, VSG). The nasal vestibule defined the anterior end of the nasal cavity, with the borders defined by soft tissue when present in individual 3DCT slices or by manually closing the nostrils when soft tissue was not present (Figure 4A-C). Posteriorly, only the nasopharyngeal lumen that was present anterior to or coincident with a line connecting the most posterior points on the right and left medial plates of the pterygoid was included in the segmented volume (Figure 4D). Comparisons between unaffected children and those diagnosed with syndromic craniosynostosis reveal potentially reduced nasal airway volumes in children diagnosed with Apert and Pfeiffer syndromes (Figs. 5 and6). Analysis of cross sectional data representing nasal airway volumes of varying groups from birth to ~30 months of age shows that children diagnosed with Apert and Pfeiffer syndromes appear to share similar nasal airway volumes with children diagnosed with Muenke syndromes and their typically developing peers at birth. Although the sample size is small, the results also indicate that children diagnosed with Crouzon syndrome may have reduced nasal airway volumes at birth. Based on this analysis using limited samples, children diagnosed with Apert and Pfeiffer syndromes may experience an early postnatal developmental divergence that results in smaller overall nasal airways within the first year of life (Fig. 5).

The distinction between true choanal atresia and more diffuse nasal airway stenosis that is often present in syndromic craniosynostosis is important for both clinical and basic research reasons. While it is paramount that researchers in the field have a clear understanding of the correct terminology in order to ensure appropriate communication and reporting, there are also potential clinical ramifications to consider. Choanal atresia in the newborn is a condition that is very amenable to early surgical intervention, which can often obviate the need for tracheostomy, prolonged NICU hospitalization, and continued respiratory monitoring. Nasal airway obstruction in the newborn with syndromic craniosynostosis may not be as readily surgically correctable in early life. Incorrect terminology may lead a surgeon down an errant pathway and may lead the child's family to have unrealistic expectations. Knowledge of associations between craniosynostosis and choanal atresia will require development of standards of diagnosis and application of those standards.

Mouse models have been developed for a number of craniosynostosis syndromes that replicate the genetic cause as well as the skeletal and soft tissue phenotypes seen in human patients, including midfacial hypoplasia [START_REF] Chen | A Ser250Trp substitution in mouse fibroblast growth factor receptor 2 (Fgfr2) results in craniosynostosis[END_REF][START_REF] Holmes | Mouse models of Apert syndrome[END_REF][START_REF] Mai | The missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc signaling[END_REF][START_REF] Twigg | Skeletal analysis of the Fgfr3P244R mouse, a genetic model for the Muenke craniosynostosis syndrome[END_REF][START_REF] Wang | Abnormalities in cartilage and bone development in the Apert syndrome FGFR2+/S252W mouse[END_REF][START_REF] Zhou | A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures[END_REF][START_REF] Eswarakumar | A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis[END_REF] . Several of these models have also been utilized to investigate nasal airway volumes. A recent study of the soft tissue phenotype of the Fgfr2c +/C342Y Crouzon/Pfeiffer syndrome mouse model found a significant reduction in nasal airway volumes in littermates carrying this mutation relative to unaffected littermates [START_REF] Martínez-Abadías | Tissue-specific responses to aberrant FGF signaling in complex head phenotypes[END_REF] . The C342Y mutation is equivalent to the most common mutation associated with human patients diagnosed with Crouzon syndrome. The human Crouzon syndrome phenotype has been associated with a number of craniofacial dysmorphologies related to both hard and soft tissues, such as premature closure of the coronal suture, midfacial hypoplasia/retrusion, and alterations to nasopharyngeal morphology [START_REF] Cohen | Craniosynostosis: Diagnosis, Evaluation, and Management[END_REF][START_REF] Johnson | Craniosynostosis[END_REF] . Skeletal phenotypic correspondences between human patients with Crouzon syndrome and the Fgfr2c +/C342Y mouse model of Crouzon syndrome have been demonstrated, and this mouse model also mimics the altered human nasopharyngeal phenotype [START_REF] Martínez-Abadías | Tissue-specific responses to aberrant FGF signaling in complex head phenotypes[END_REF] . At P0 (day of birth), heterozygous Fgfr2c +/C342Y littermates exhibited a statistically significant restriction in nasal airway volume (2.81 ± 0.17 mm 3 ), as compared to their unaffected littermates (3.28 ± 0.13 mm 3 , p = 0.012) [START_REF] Martínez-Abadías | Tissue-specific responses to aberrant FGF signaling in complex head phenotypes[END_REF] . However, choanal atresia was not reported in any of the mice studied. As of this date, there have been no published reports of definitive choanal atresia in any mouse models of syndromic craniosynostosis.

While murine data provide valuable information about the molecular and developmental mechanisms that produce the choanae, significant differences in human and murine craniofacial anatomy and development must be taken into consideration when evaluating the comorbidity of choanal atresia and craniosynostosis using cross-species comparisons. Due to the rostral-caudal elongation of the murine premaxillae, maxillae, palatine bones, and the soft palate, different osteological and soft tissue boundaries define the murine choanae relative to humans. In humans, choanal atresia has been attributed to a combination of a thickening of the posterior vomer with medialization of the pterygoid plates of the sphenoid.

Although useful murine models of choanal atresia have recently been produced and will be critical to determining the molecular and developmental basis of choanal atresia [START_REF] Kurosaka | Rdh10 loss-of-function and perturbed retinoid signaling underlies the etiology of choanal atresia[END_REF] , speciesspecific differences including the anatomical separation of the vomer and pterygoid plates along the rostro-caudal axis in mice suggests an alternate structural foundation for murine choanal atresia (Fig. 7). While mouse models are an excellent tool for understanding the etiology of human craniofacial disorders like craniosynostosis, given the tremendous number of genetic mutations implicated in craniosynostosis conditions, each model can represent only a single development pathway to the craniosynostosis phenotype. We propose that there are potentially as many ways to produce choanal atresia.

The significant correlation between specific craniosynostosis syndromes and reduced nasal airway volume in mouse models for craniosynostosis and human pediatric patients indicates comorbidity of choanal and nasopharyngeal dysmorphologies and craniosynostosis conditions. Genetic, developmental and epidemiologic sources of these interactions are areas particularly worthy of further research. Mid-sagittal section of adult human showing the position of the choanae relative to the human nasal, oral, and pharyngeal airways. 3DCT reconstruction of a typically developing child (left) showing superimposed segmentations of skin surface (beige), brain surface (grey) and upper airway lumen (blue). At right are "virtual endocasts" of the nasopharynx of a child with Apert syndrome (pink, at left) and a typically developing child (maroon, at right) as segmented from high resolution 3DCT. Superimposition of the two virtual endocasts (center) shows local areas of greatest shape difference. This comparison is not a statistical comparison of the nasopharyngeal anatomy of patients with Apert syndrome and typically developing individuals; rather, this superimposition provides an example of how nasopharyngeal morphology of craniosynostosis patients may differ from typically developing individuals. 3DCT reconstruction of the cranium of 6 week old C57BL/6J mouse showing the bones that form the osteological borders of the choanae in the human skull: vomer (blue), basisphenoid (pink), medial pterygoid plates (red), and horizontal plates of the palatine bones (purple).

The vomer (which is ghosted in this illustration) lies deep to the maxillae and so is hidden in an inferior view. The presphenoid is shown in green. Note the anatomical separation of these bones compared to the human skull in Figure 1. The black arrow indicates the position of the choanae in mice at the soft tissue intersection of the posterior nasal cavity and nasopharynx. Case reports that explicitly diagnose syndromic craniosynostosis and choanal atresia. NR=not reported.

Gene

Figure 1 .

 1 Figure 1.3DCT reconstruction of the cranium of a typically developing child viewed from below showing the osteological borders of the choanae: vomer (blue), sphenoid body (pink), medial pterygoid plates (red), and horizontal plates of the palatine bones (purple).

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.Formation of the secondary palate and choanae. Inferior view of the forming palate showing (left) vertically oriented palatal shelves, (center) the palatal shelves as they rotate downward into a horizontal position and begin to approximate one another to form the primary choanae, and (right) fused palatal shelves in their final orientation, with the incisive foramen at the intersection of the primary and secondary palate and the secondary, or definitive, choanal openings at the posterior end of the palate. (Adapted from 3)

Figure 4 .

 4 Figure 4.Nasal airway segmented from CT image of a typically developing child at 10 months, as an example of how the nasal airway volume data presented in Figure5were collected. Top Left) Axial CT image at the level of the orbits indicating area of close-up (red box) for three additional anatomical levels including: Top Right) the nasal cavity (red) with soft tissue of the nose bordering the anterior nares at the level of the maxillary sinuses; Bottom Left) midnares level with partial soft tissue border of the anterior nares; and Bottom Right) at the level of the alveolar processes of the maxillae showing the nasopharyngeal lumen (red) anterior to a line (blue) connecting the most posterior points on the medial plates of the pterygoid bone.

Figure 5 .

 5 Figure 5. Scatterplot of total nasal airway volume and age (in months) of individuals diagnosed with various craniosynostosis syndromes and typically developing individuals. Sample sizes are as follows: Apert (n = 13), Crouzon (n = 10), Muenke (n = 5), Pfeiffer (n = 5), unaffected (n = 39). Lines representing the results of regression analysis showing the relationship between age and total nasal airway volume (including the ethmoidal air cells) for each group. It is important to note that this plot and the regression lines estimated from the cross-sectional data are used to demonstrate the variation in nasal airway volumes among craniosynostosis syndromes. As the nasal airway volume data are based on cross-sectional datasets for each diagnostic category, these regression lines do not necessarily indicate growth patterns or growth trajectories.

Figure 6 .

 6 Figure 6.

Figure 7 .

 7 Figure 7.

  ). Table 1 lists published case reports that have explicitly reported choanal atresia in patients diagnosed with syndromic craniosynostosis. Craniosynostosis cases reporting only choanal stenosis are not included.

Table 1

 1

Plast Reconstr Surg. Author manuscript; available in PMC 2019 January 01.

Acknowledgments

The authors thank members of the Bernstein, Jabs, and Richtsmeier labs for ongoing discussions about the development, anatomy and dysmorphology of the choanae that informed this study, including Susan Motch Perrine, Kevin Flaherty, Kazuhiko Kawasaki, Mizuho Kawasaki, Greg Holmes, James Azzi, and Sameep Kadakia. Use of the CT images was approved by the Institutional Review Boards of the Pennsylvania State University and the participating institutions and the images were acquired in accordance with institutional guidelines. All collected images were anonymized and no information other than sex, age at the time of the CT exam, and causative mutation were available. Research presented here was funded in part by the National Institutes of Health (NIDCR, NICHHD and ARRA) [R01-DE018500, R01-DE018500-S1, R01-DE022988, P01HD078233] and the National Science Foundation [BCS-0725227].

Author Manuscript


Impact of sampling strategies and reconstruction protocols in nasal airflow simulations in fossil hominins

Andrej Evteev, Yann Heuzé

Impact of sampling strategies and reconstruction protocols in nasal airflow simulations in fossil hominins

To whom correspondence should be addressed. Email: evteandr@gmail.com

In their study, de Azevedo et al. ( 1) employ a sample of 12 individuals from Argentina of Chinese, Japanese, and Korean origin [northeastern Asians (NEA)] as representative of cold-adapted populations. However, all previous literature on the subject shows that the craniofacial morphology of these populations does not exhibit features adapted to a cold climate (e.g., refs. 2, 3). In fact, the climate of the most populated parts of China and Japan is temperate and cannot be referred to as "cold." As stated by the authors, Arctic populations drive climate-morphology correlations in many studies (4), including their own (ref. 1, p. 4).

Additionally, it is surprising that no differences in the morphology of the anterior nasal region were found between the NEA and southwestern Europeans (SWE) (figure 1 of ref. [START_REF] De Azevedo | Nasal airflow simulations suggest convergent adaptation in Neanderthals and modern humans[END_REF], despite wellestablished differences in craniofacial features between those groups (e.g., ref. [START_REF] Howells | Skull Shapes and the Map: Craniometric Analyses in the Dispersion of Modern Homo[END_REF]. This leads to a general concern that the sample is poorly described (no sex and age distributions). More importantly, no criteria are given for choosing the two individuals who were used in the subsequent computational fluid dynamics (CFD) model (ref. 1, p. 5). Consequently, this produces the main, in our opinion, issue of this study: a lack of knowledge about the factors contributing to differences in the soft tissue airway shape of those two individuals. Indeed, the nasal mucosa is an erectile tissue; its state of congestion is affected by numerous factors (e.g., nasal cycle). Therefore, the nasal airway's shape and size can fluctuate rapidly and to a very substantial degree (e.g., ref. [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF]. That is the reason why the criteria for choosing the SWE and NEA individuals used in the CFD model should be stated in detail. As a result, the huge differences reported in the CFD results between these two individuals (figure 2 ) between the bone and soft tissue of the nasal cavity is too weak to predict soft tissue morphology from bone morphology. Third, it has been shown that there is no correlation between the shape of the nasal aperture and that of the choanae in humans [START_REF] Bastir | Cranial airways and the integration between the inner and outer facial skeleton in humans[END_REF]. Ignoring this study, the authors performed their own study (ref. 1, p. 2 and SI Appendix, pp. 3-7), where they analyzed representatives of different families of primates together. While some correlations were found (figures S3-S5 of ref. [START_REF] De Azevedo | Nasal airflow simulations suggest convergent adaptation in Neanderthals and modern humans[END_REF], it remains unclear whether the pattern of association would remain the same, vary, or be absent at a lower taxonomic level. Several reconstructions of Neanderthal nasal

  Andrej A. Evteev a,1 and Yann Heuzé b a Anuchin's Research Institute and Museum of Anthropology, Lomonosov Moscow State University, 125009 Moscow, Russia b CNRS, University of Bordeaux, Ministère de la Culture, De la Préhistoire à l'Actuel: Culture, Environnement et Anthropologie (PACEA), UMR 5199, 33615 Pessac Cedex, France 1

  of ref. 1) are highly questionable. Finally, how the nasal cavity and mucosa are modeled in the Neanderthal specimen also raises several questions. First, one SWE individual was used as the starting shape for the model (ref. 1, p. 5), and the results for the SWE and Neanderthal individuals were similar (figure 1 of ref. 1). What would have been the results if one NEA individual had been used instead? Second, the association detected by the authors (figure S7 of ref. 1

Author contributions: A.A.E. and Y.H. designed research and wrote the paper.

The authors declare no conflict of interest.


Yann Heuzé
email: yann.heuze@u-bordeaux.fr

What does nasal cavity size tell us about functional nasal airways? Qu'est-ce que la taille de la cavité nasale nous dit sur les voies aériennes nasales ?

Keywords: Nasal cavity vs. nasal airways air conditioning, respiratory energetics, computed tomography, in vivo conditionnement de l'air, dimension énergétique de la respiration, tomographie, in vivo

Studies on dry human skulls have shown that nasal cavity (NC) morphology varies according to ecogeographic factors. These findings have been used by some authors to interpret the facial morphology of Neanderthals. However, respiratory and air conditioning functions are primarily carried out by the nasal airways (NA) which are delimited by mucosa. The aims of this study were to test whether: 1) NC volume (V) and surface area to volume ratio (SA/V) are proportional to NA counterparts;

2) males NC and NA measurements are larger than those of females; 3) the centroid size (CS) of a set of landmarks measured on NC provides a reliable proxy for NC V. Head CT images of adults (N = 30) who consulted at the University Hospital of Bordeaux were retrospectively selected. NA were defined by segmenting the lumen corresponding to the functional volume. NC was defined by adding to NA the soft tissues delimited by the bones forming NC. The coordinates of 16 landmarks measured on NC bones were recorded. A rather low correlation was found between NA and NC V while NA SA/V and NC SA/V were not correlated. No significant differences between male and female NA and NC measurements were found. A rather low correlation was found between NC V and NC CS. If these preliminary results were to be confirmed by future studies, results using NC as a proxy for NA focusing on air conditioning and respiratory energetics might need to be re-interpreted.

Résumé

Plusieurs études sur des crânes secs humains ont révélé que la morphologie de la cavité nasale (NC) varie selon des facteurs écogéographiques. Ces résultats ont été utilisés par certains auteurs pour interpréter la morphologie faciale des Néandertaliens. Cependant, les fonctions respiratoires et le conditionnement de l'air sont assurés en premier lieu par les voies aériennes nasales (NA) qui sont délimitées par une muqueuse. Les buts de cette étude étaient de tester si : 1) le volume (V) et le rapport de l'aire surfacique sur le volume (NA/V) de NC sont proportionnels à ceux de NA ; 2) NC et NA sont plus grandes chez les hommes ; 3) la taille centroïde (CS) représente un indicateur fiable du V de NC. Des images tomographiques d'individus adultes (N = 30) du CHU de Bordeaux ont été sélectionnées de manière rétrospective. NA étaient définies par la segmentation du lumen correspondant au volume fonctionnel.

NC était définie en ajoutant à NA les tissus mous délimités par les os formant NC. Les coordonnées de 16 landmarks mesurés sur les os de NC ont été enregistrées. Une faible corrélation a été trouvée entre les V de NA et NC tandis qu'aucune n'a été identifiée entre NA SA/V et NC SA/V. Aucune différence significative entre hommes et femmes n'a été trouvée. Une corrélation relativement faible a été identifiée entre NC V et NC CS. Si ces résultats préliminaires venaient à être confirmés par de futures études, les résultats utilisant NC comme un proxy pour NA pour l'étude des capacités respiratoires et du conditionnement de l'air devraient être réinterprétés.

INTRODUCTION

Several studies on dry skull materials of different human populations from different latitudes have shown that nasal cavity (NC) morphology varies according to ecogeographic factors, such as temperature and humidity. Historically, the relationships between climate and NC size and proportions was established by studies focusing primarily on the dimensions of the nose and the nasal aperture [START_REF] Thomas | Man's nasal index in relation to certain climatic conditions[END_REF][START_REF] Davies | A re-survey of the morphology of the nose in relation to climate[END_REF][START_REF] Weiner | Nose shape and climate[END_REF][START_REF] Carey | Human nasal protrusion, latitude, and climate[END_REF][START_REF] Franciscus | Variation in human nasal height and breadth[END_REF][START_REF] Roseman | Detecting interregionally diversifying natural selection on modern human cranial form by using matched molecular and morphometric data[END_REF][START_REF] Hubbe | Climate signatures in the morphological differentiation of worldwide modern human populations[END_REF].

Later on, studies taking into account the entire NC confirmed this relationship, particularly for populations living in extremely cold climates [START_REF] Noback | Climate-related variation of the human nasal cavity[END_REF][START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF][START_REF] Evteev | Extreme climate, rather than population history, explains mid-facial morphology of Northern Asians[END_REF][START_REF] Fukase | Geographic variation in nasal cavity form among three human groups from the Japanese Archipelago: Ecogeographic and functional implications[END_REF]. Populations living in cold climates tend to display a narrower nasal aperture, sagittally enlarged NC, and increased surface-area-to-volume ratio (SA/V) of NC, resulting in a greater air conditioning capacity [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF][START_REF] Churchill | Morphological variation and airflow dynamics in the human nose[END_REF][START_REF] Doorly | Nasal architecture: form and flow[END_REF][START_REF] Holton | Climatic adaptation and Neandertal facial evolution: a comment on Rae et al[END_REF][START_REF] Holton | The morphological interaction between the nasal cavity and maxillary sinuses in living humans[END_REF][START_REF] Maddux | Absolute humidity and the human nose: A reanalysis of climate zones and their influence on nasal form and function[END_REF]. On the other hand, a recent paper by Maddux et al. [START_REF] Maddux | Ecogeographic variation across morphofunctional units of the human nose[END_REF] indicates that the internal nasal fossa is the only component of the nasal complex (i.e., external pyramid, nasal aperture, internal nasal fossa, and nasopharynx) which displays an ecogeographic pattern of variation consistent with adaptation to climatic conditions. Because European Neanderthals lived during periods characterized by glacial conditions, many aspects of their morphology, including facial skeletal traits (e.g. nasal cavity and paranasal sinuses), have been interpreted as adapted to cold climate [e.g. 18]. However, genetic drift has been shown to be able to account for several of these traits [e.g. 19]. Consequently, the meaning of Neanderthal skeletal morphology is still debated.

The NC is a negative space delimited by bones housing the nasal airways (NA). As such, NC is the gateway to the respiratory system [START_REF] Enlow | Facial Growth 3 Sub edition[END_REF] and indirectly participates in inspired and expired air conditioning. NC displays a variable and complex morphology based on the shape and size of the aspects of the bones forming that negative space; the maxilla, nasal, palatine, vomer, sphenoid, frontal, ethmoid, and lacrimal bones. The superior, middle, and lower nasal turbinates or conchae contribute to the complexity of NC morphology. The quantity of inspired air depends on NC size and shape and particularly of its height and width, especially at its entrance and exit points (i.e. piriform aperture and the choanae, respectively) [START_REF] Swift | Access of air to the respiratory tract[END_REF][START_REF] Bastir | Cranial airways and the integration between the inner and outer facial skeleton in humans[END_REF]. Consequently, NC morphology likely influences respiratory energetics. The effectiveness of air conditioning is also related, at least partially, to NC morphology, particularly to NC length (e.g. [START_REF] Noback | Climate-related variation of the human nasal cavity[END_REF]) and SA/V (e.g. [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF]).

However, the functional negative space primarily responsible for air conditioning is represented by the NA. NA are delimited by mucosa and housed within the NC. Because of its dense vascularization, NA mucosa experiences variation in thickness depending on several possibly interrelated factors, including blood pressure, temperature, humidity, and nasal cycle [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF][START_REF] Cauna | Blood and nerve supply of the nasal lining[END_REF][START_REF] White | Model demonstrates functional purpose of the nasal cycle[END_REF]. This results in fluctuations in the level of congestion (reduced volume), or its opposite condition, decongestion, exhibited by NA. Obviously, only studies based on living individuals allow NA morphology to be analyzed and quantified, and computed tomography (CT) images offer appropriate material for that purpose. Very few studies quantifying NA morphology have been published to date. One such study is that of Yokley [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF] in which the author uses a 2D approach for in vivo CT images to compute the ratio between the perimeter and area of one coronal section through the nasal passages. These measurements were done twice per specimen, one with NA partially congested by the nasal mucosa and another with nasal airways fully decongested after virtually removing the nasal mucosa. Only the measurements of the fully decongested NA were found to correlate with ecogeographic variables. However, the results obtained with fully decongested NA are somewhat artificial since they are based on the hypothesis that the thickness of the mucosa is null, which effectively results in measuring NC.

Therefore, the following question still remains: what does NC size (i.e. volume, V) tell us about functional NA? In other words, what are the differences between NC and NA size when the mucosa is taken into account? The aim of the present study is to quantify NC size and air conditioning potential (i.e. SA/V) and to compare them with those of NA. Since NC volume is often approximated by the centroid size (square root of the sum of squared distances of a set of landmarks from their centroid, CS) of a set of anatomical landmarks measured on the bones forming the NC, we also explore whether CS is a reliable proxy for NC volume. Three working hypotheses were tested throughout the present study: (H1) NC V, surface area (SA), and SA/V are proportional to NA counterparts; (H2) because of larger body and lean mass, males display larger NC and NA than females; (H3) CS of a set of anatomical landmarks measured on the bones forming the NC provides a reliable proxy for NC volume.

MATERIALS AND METHODS

Head CT angiogram images of adult patients who consulted at the emergency room of the University Hospital of Bordeaux have been selected for the purpose of this study (slice thickness: 0.625mm). The following patients were systematically excluded: those younger than 18 years, older than 59 years, with major facial skeletal dysmorphology, under respiratory assistance, or with obstructed nasal airways.

Patients imaged with their mouth open were also excluded to avoid potential bias linked to mouth breathing. Fifteen men and fifteen women have been included in our sample (mean age = 31.8 years; SD = 11.0 years). There was no significant age differences between males and females (t = -0.0652, p = 0.9502). All images were collected retrospectively and anonymized. Only the sex and exact age at exam were kept. This study has been reviewed and approved by the Univ. Bordeaux IRB (Comité de Protection des Personnes Sud-Ouest et Outre Mer III).

First, the NA were defined by segmenting the lumen corresponding to the airway's functional volume and were delimited anteriorly by the nasal aperture and posteriorly by the choanae (Fig. 1A,1C).

Because of the difficulty in objectively determining where the NA end and where the ethmoidal cells begin, the latter were included in what is defined as NA in the present study. The NC was defined by segmenting both the lumen (i.e., NA) and the soft tissues covering the bones forming the NC (from anterior nasal aperture to choanae) (Fig. 1B,1D). Though segmenting nasal conchae can sometimes be challenging (e.g. [START_REF] Uosyte | Effects of fluid and computed tomographic technical factors on conspicuity of canine and feline nasal turbinates[END_REF]), they were segmented as bone and not included in the computation of NC measurements. NA and NC were segmented in the transverse plane and inspected in the coronal and sagittal planes (Fig. 1). Both NC and NA were closed on each transverse slice anteriorly by a line joining the two aspects of the maxilla or the nasal bones forming the nasal aperture, and posteriorly by a line joining the distal walls of the choanae formed by the sphenoid and passing through the most posterior aspect of the vomer (Fig. 1). For NA and NC, both left and right sides were considered to avoid potential effects of nasal cycle. Nasal cycle is characterized by recurrent variation in air mass-flow partitioning between the right and left airways, which alternatively display different level of mucosa congestion, enabling each passageway to alternatively take turns in either predominantly undertaking the airconditioning or mucus clearance roles [START_REF] White | Model demonstrates functional purpose of the nasal cycle[END_REF]. Because of the central aspect of the segmentation process in the present study, the repeatability of the protocol was quantified by measuring five individuals twice (one set of measure at the beginning of the data collection, one set in the end). The results are presented in table 1 and show that the intra-observer error is negligible.

Additionally, 16 three dimensional landmarks were measured on bone (Fig. 2). The landmarks' 3D coordinates were then analyzed with geometric morphometrics using general Procrustes superimposition [START_REF] Rohlf | Extensions of the Procrustes method for the optimal superimposition of landmarks[END_REF][START_REF] Dryden | Statistical Shape Analysis[END_REF]. Centroid size (CS, the square root of the summed squared distances of each landmark to the centroid) was used as a proxy for NC size.

Next, because it has been shown that airflow in the lower and middle meatus account for over 80% (50% and 30% respectively) of the total nasal airflow [START_REF] Xiong | Computational fluid dynamics simulation of airflow in the normal nasal cavity and paranasal sinuses[END_REF], hormion and the anteriormost point on the left and right nasomaxillary sutures were selected to define a plane that was subsequently used to separate the upper meatus from the middle and lower meatus. Only the latter portion was then studied. Though this plane might not be ideal, it was the one among the several planes tested passing through the measured landmarks, which gave the most satisfactory results for the separation of the upper meatus from the middle and lower meatus.

The ratio SA/V was computed for both the NC and NA as it represents a relevant proxy for air conditioning potential.

Finally, to broadly describe NC relative dimensions, linear measurements accounting for NC length (from nasomaxillary suture, left to choanal roof, left) and NC width (mean between inter-lower turbinate base and inter-alare distances) were recorded (Fig. 2). NC height was measured from, superiorly, the anteriormost point of the inferior surface of the cribriform plate taken on a slice parallel to the mid sagittal plane (passing through nasale, anterior nasal spine, posterior nasal spine), to, inferiorly, the perpendicular projection of the superior point onto the nasal surface of the maxilla. This slice was the closest to the midsagittal plane for which the vomer was not visible anymore to avoid artificial reduction of NC height (Fig. 2). Larger NC height and width are expected to facilitate volumetric intake of air, while NC length has no influence. However larger NC length is expected to facilitate air conditioning by increasing air resident time.

T-tests with Monte Carlo permutations (n = 9,999) and Pearson's correlation coefficients with Monte Carlo permutations (n = 9,999) were used to test our hypotheses. For these tasks, the software package PAST was used [START_REF] Hammer | PAST: Paleontological Statistics Software Package for Education and Data Analysis[END_REF].

RESULTS

H1: NC V, SA, and SA/V are proportional to NA counterparts When considering the entire NC and NA, including the ethmoidal cells, there was no significant correlation between NA V and NC V (R² = 0.0445, p = 0.2625), though there was a significant correlation between NA SA and NC SA (R² = 0.2305, p = 0.0075) (Table 2). There was no significant correlation between NA SA/V and NC SA/V (R² = 0.0065, p = 0.7066) (Table 2). NA SA/V was characterized by a higher dispersion (coefficient of variation: cv = 0.223) than NC SA/V (cv = 0.144).

When only considering the lower two thirds of NA and NC, a significant correlation, though relatively low, was found between NA V and NC V (R² = 0.1408, p = 0.0349) and another between NA SA and NC SA (R² = 0.7066, p = 0.0001) (Table 2). There was no significant correlation between NA SA/V and NC SA/V (R² = 0.0078, p = 0.6234) (Table 2). NA SA/V was characterized by a higher dispersion (cv = 0.313) than NC SA/V (cv = 0.131). H2: Males display larger NC and NA than females When considering the entire NA and NC, no significant differences were found between males and females for NA V (t = 1.2721, p = 0.2144), NA SA (t = -0.1336, p = 0.8982), NA SA/V (t = -2.0569, p = 0.0510), NC V (t = -1.8106, p = 0.0792), NC SA (t = -0.1429, p = 0.2599), nor NC SA/V (t = 0.1149, p = 0.9613) (Table 3). When only considering the lower two thirds of NA and NC, there was no significant differences between male and female NA V (t = 1.1018, p = 0.2736), NA SA (t = -0.1778, p = 0.8584), NA SA/V (t = -1.4302, p = 0.1575), NC V (t = -1.4307, p = 0.1656), NC SA (t = -0.5453, p = 0.5925), or NC SA/V (t =0.9974, p = 0.3319) (Table 3). While males displayed overall larger measurements than female, females displayed larger NA V, either considering the entire NA or only the lower two thirds.

Though males displayed overall larger NC linear measurements, no significant differences between male and female NC height (t = 1.2981, p = 0.2024), NC width (t = 0.8589, p = 0.3950), nor NC length (t = 0.9827, p = 0.3301) were found (Table 4).

H3: CS of NC provides a reliable proxy for NC volume

There was a significant, though rather low, correlation between NC V and NC CS, either when considering the entire NC (R² = 0.3597, p = 0.0005) or only NC lower two thirds (R² = 0.1944, p = 0.0147). Male NC CS was significantly larger than female NC CS only when considering the entire NC (t = 2.5138, p = 0.0180).

When considering the lower two thirds of NC, there was no significant differences between male and female NC CS (t = 1.9556, p = 0.0606).

DISCUSSION

Based on our results, H1, stating that NC V, SA, and SA/V are proportional to NA counterparts, could only be supported with some limitations. Indeed, only the volume of the lower two thirds of NC was significantly correlated with that of NA. However this correlation appeared relatively low (R² = 0.1408).

Though the correlation was high for SA between NA and NC, this was not the case for SA/V which is commonly used as a proxy for air conditioning potential. Consequently, it might be more appropriate to consider only the lower and middle meatus when measuring NC. This is consistent with the fact that NA lower two thirds account for over 80% of the total nasal airflow [START_REF] Xiong | Computational fluid dynamics simulation of airflow in the normal nasal cavity and paranasal sinuses[END_REF]. However, it appeared that interpolation made on air conditioning potential on the basis of NC SA/V could be problematic.

This study revealed no significant differences between male and female NA V, NC V, NA SA, NC SA, NA SA/V, or NC SA/V, either considered as a whole or for only the lower two-thirds. Consequently, H2, stating that males display larger NC and NA than females, should be rejected. Because NC height and width primarily constraint volumetric intake of air, both were expected to be significantly larger in males.

However, none was significantly larger in males than in females. Overall, these results bring little support to the hypothesis that, due to larger body size and lean mass, male NC and NA are larger than female NC and NA. However, these results are based on a rather small sample size (n = 30) and it is expected that with a larger sample some of these differences might surpass the significance threshold.

The 16 anatomical landmarks measured in the present study on NC correspond to the measurable landmarks on CT images from a list of landmarks classically measured on dry skulls in comparable studies [e.g. [START_REF] Noback | Climate-related variation of the human nasal cavity[END_REF][START_REF] Evteev | Extreme climate, rather than population history, explains mid-facial morphology of Northern Asians[END_REF][START_REF] Fukase | Geographic variation in nasal cavity form among three human groups from the Japanese Archipelago: Ecogeographic and functional implications[END_REF][START_REF] Bastir | Cranial airways and the integration between the inner and outer facial skeleton in humans[END_REF]. Based on our results, the CS of such a landmark configuration appears to be a relatively poor proxy for NC volume especially when considering the lower two thirds of NC (R² = 0.1944). Thus, H3, stating that CS of NC provides a reliable proxy for NC volume, should be rejected. It is unlikely that the rather complex geometry of NC could be captured in an optimal manner by these external landmarks that might over-simplify NC complex geometric properties. Consequently, the measure of NC volume might be the only reliable way to quantify NC size.

NA appears much more variable than NC. One obvious explanatory factor of this inter-and intraindividual variation is the mucosa itself, which varies in volume and consequently contributes to variation in NA volume as well. In contrast, for the same individual, the NC volume remains constant.

This study attempted to control for a maximum of factors influencing mucosa congestion/decongestion state. First, only patients breathing by the nose, without respiratory assistance, and with their mouth closed during the CT exam were selected. Second, a relative rest state was assumed at the time of imaging (i.e., no engagement in major physical activities), as well as relatively homogeneous environmental conditions as temperature and humidity levels were controlled by air conditioning. Third, nasal cycle was taken into account by measuring the right and left NA rather than just one side. Since the present study focused on rest state condition, considering the fully decongested NA did not appear as a relevant approach. Note that during physical exercise, the nasal mucosa decongests almost to the same level as after using topical decongestant [START_REF] Hilberg | Objective measurement of nasal airway dimensions using acoustic rhinometry: methodological and clinical aspects[END_REF], though a significant part of respiration is carried out by the mouth. The level of stress experienced by the patients could, however, not be controlled. Despite all these precautions taken, NA SA/V was twice to thrice more variable than NC SA/V. This result is in agreement with that obtained by Yokley [START_REF] Yokley | Ecogeographic variation in human nasal passages[END_REF] in 2D showing that, when the mucosa was taken into account (i.e., not fully decongested), variation in SA/V was too substantial to observe a significant difference for SA/V between individuals of European descent and African descent.

Another explanatory factor to the overall low correlation existing between NA and NC measurements is the anatomical complexity of the nasal conchae and the acknowledged difficulty to segment them on CT images. This difficulty is linked to the complex geometry of the nasal conchae and the surrounding soft tissues and fluids [START_REF] Uosyte | Effects of fluid and computed tomographic technical factors on conspicuity of canine and feline nasal turbinates[END_REF]. While the nasal conchae were segmented as bone and consequently not taken into consideration when computing NC measurements, it is possible that for some individuals an imperfect segmentation resulted in the inclusion of small parts of the nasal conchae in NC measurements. The fact that males displayed larger mean NC volumes but smaller mean NA volumes compared to females (Tables 3) could suggest that males have larger nasal conchae potentially associated with larger mucosa volumes.

The results of the present preliminary study seem to challenge the validity of the often assumed or implied correlation between NA and NC size/volume. Because of its relatively small sample size and its methodological limitations (e.g. segmentation of the conchae, mucosa thickness variation), these preliminary results remain to be validated by future studies that will provide a more robust description of the relationship between NA and NC. If these preliminary results were to be confirmed by future studies, results produced by studies based on dry skull materials focusing on air conditioning and respiratory energetics using NC as a proxy for NA might need to be re-interpreted. Studies on these topics including fossils such as Neanderthals, for which the NC is rarely fully conserved, are implicated as well. These future studies will need to use larger in vivo samples, as well as possibly those originating from different regions of the world, to provide more in depth results pertaining to the nature of the relationship between NA and NC morphological variation and co-variation and their implications for air conditioning and respiratory energetics. One key aspect for this future work will be to use imaging parameters optimized for nasal conchae visualization and segmentation to investigate the potential role of the nasal conchae and the overlying mucosa in the differences observed between NC and NA measurements.

Fig. 1

 1 Fig. 1 Segmentation of the nasal airways (NA) (A) and nasal cavity (NC) (B). NA and NC are delimited

Fig. 2

 2 Fig. 2 Landmarks (n = 16) were measured on bone to compute the centroid size of the nasal cavity (NC)

Fig. 1 20Fig. 2

 12 Fig. 1

Table 3

 3 Nasal airways (NA) and nasal cavity (NC) volume (V), surface area (SA), and surface area to volume ratio (SA/V) by sex a,b / Volume (V), aire surfacique (SA) et ratio aire surfacique sur volume (SA/V) des voies aériennes nasales (NA) et de la cavité nasale (NC) selon le sexe. Values are means in cm3 for volumes and mm² for surface areas / Les valeurs correspondent à des moyennes en cm3 pour les volumes et en mm² pour les aires surfaciques.Values are Student t-values (t) and significance after Monte Carlo permutations (p, in parenthesis) / Les valeurs correspondent à des valeurs de test t de Student (t) et à leur significativité après simulations de Monte Carlo (p, entre parenthèses).

		NA V	NC V	NA SA	NC SA	NA SA/V	NC SA/V
	Entire nasal airways (NA) and nasal cavity (NC)
	M	22.55	63.38	24.86	21.5	1.17E-03	3.40E-04
	F	25.37	59.21	24.70	20.20	9.98E-04	3.42E-04
	M vs. F 1.27 (0.2144)	-1.81 (0.0792) -0.13 (0.8982) -0.14 (0.2599) -2.06 (0.0510) 0.11 (0.9613)
	Lower two-thirds of nasal airways (NA) and nasal cavity (NC)
	M	9.90	32.85	10.82	10.69	1.20E-03	3.27E-04
	F	11.33	30.39	10.69	10.36	1.02E-03	3.43E-04
	M vs. F 1.10 (0.2736) -1.4307 (0.1656) -0.18 (0.8584) -0.55 (0.5925) -1.43 (0.1575) 1.00 (0.3319)

a b

Table 4

 4 Nasal cavity linear measurements by sex a,b / Mesures linéaires de la cavité nasale selon le sexe Les valeurs correspondent à des moyennes exprimées en mm.

		NC height	NC width	NC lenght
	M	49.2	25.8	71.2
	F	47.8	25.3	69.8
	M vs. F	1.2981 (0.2024) 0.8589 (0.3950) 0.9827 (0.3301)
	b Values are Student t-values (t) and significance after Monte Carlo permutations (p, in parenthesis) / Les
	valeurs correspondent à des valeurs de test t de Student (t) et à leur significativité après simulations de
	Monte Carlo (p, entre parenthèses).

a Values are means in mm /

ACKNOWLEDGEMENTS Y.H. is supported by the research program LaScArBx from the French National Research Agency (ANR) and its program "Investissements d'avenir" [ANR-10-LABX-52]. The author is grateful to Drs. Andrej Evteev, Sébastien Villotte, Lauren Butaric, Christina Nicholas, Jessica Joganic and Christine Couture for interesting discussions on previous versions of this work. Comments from the two reviewers (Drs. Scott

YH is supported by the research program LaScArBx from the French National Research Agency (ANR) and its program "Investissements d'avenir" [ANR-10-LABX-52]