Cholesteatoma Severely Impacts the Integrity and Bone Material Quality of the Incus.

Cholesteatoma can lead to progressive destruction of the auditory ossicles along with conductive hearing loss but precise data on the microstructural, cellular, and compositional aspects of affected ossicles are not available. Here, we obtained incus specimens from patients who had cholesteatoma with conductive hearing loss. Incudes were evaluated by micro-computed tomography, histomorphometry on undecalcified sections, quantitative backscattered electron imaging, and nanoindentation. Results were compared with two control groups taken from patients with chronic otitis media as well as from skeletally intact donors at autopsy. The porosity of incus specimens was higher in cholesteatoma than in chronic otitis media, along with a higher osteoclast surface per bone surface. Histomorphometric assessment revealed higher osteoid levels and osteocyte numbers in cholesteatoma incudes. Incudes affected by cholesteatoma also showed lower matrix mineralization compared with specimens from healthy controls and chronic otitis media. Furthermore, the modulus-to-hardness ratio was higher in cholesteatoma specimens compared with controls. Taken together, we demonstrated increased porosity along with increased osteoclast indices, impaired matrix mineralization, and altered biomechanical properties as distinct features of the incus in cholesteatoma. Based on our findings, a possible impact of impaired bone quality on conductive hearing loss should be further explored.

Internal vascular channel architecture in human auditory ossicles.

The vascular supply of the human auditory ossicles has long been of anatomical and clinical interest. While the external blood supply has been well-described, there is only limited information available regarding the internal vascular architecture of the ossicles, and there has been little comparison of this between individuals. Based on high-resolution micro-CT scans, we made reconstructions of the internal vascular channels and cavities in 12 sets of ossicles from elderly donors. Despite considerable individual variation, a common basic pattern was identified. The presence of channels within the stapes footplate was confirmed. The long process of the incus and neck of the stapes showed signs of bony erosion in all specimens examined. More severe erosion was associated with interruption of some or all of the main internal vascular channels which normally pass down the incudal long process; internal excavation of the proximal process could interrupt vascular channels in ossicles which did not appear to be badly damaged from exterior inspection. An awareness of this possibility may be helpful for surgical procedures that compromise the mucosal blood supply. We also calculated ossicular densities, finding that the malleus tends to be denser than the incus. This is mainly due to a lower proportion of vascular channels and cavities within the malleus.

Navigational guidelines and positional relationships of the human auditory ossicles from three-dimensional topography for ensuring safe and effective malleostapedotomy : Stereotactic topography of the auditory ossicles and its clinical implication.

PURPOSE: The aims of the present study were to identify detailed positional relationship between the auditory ossicles and to provide theoretic navigational guidelines for optimal prosthesis adaptation and effective malleostapedotomy. METHODS: Fifty sides of the temporal bone from donated cadavers were scanned by MicroCT and the malleus, incus, stapes and tympanic membrane were materialized three dimensionally using computer software. Dimensions between the auditory ossicles closely related to malleostapedotomy were measured twice. RESULTS: The grip site of malleus handle was mean 1.8 mm superior and mean 1.3 mm anterior, and linear distance between the grip site of malleus handle and the footplate of the stapes was mean 6.5 mm. The stapes was not parallel to the tympanic membrane and rotated mean 10.7° posteriorly relative to the tympanic membrane. CONCLUSION: Surgeons should start with at least 8.75 mm prosthesis to cover the upper limits of potential anatomy and then trim down to the individualization to the case. The ideal loop morphology has to be oval shape more than 1.4 mm in the long diameter and 1.0 mm in the short diameter. The wire of the prosthesis has to be bended at the two points: about 10° anteriorly at the most proximal point of the wire and about 50° superiorly at the stapes head point.

[Numerical study on the effect of middle ear malformations on energy absorbance].

In order to study the effect of middle ear malformations on energy absorbance, we constructed a mechanical model that can simulate the energy absorbance of the human ear based on our previous human ear finite element model. The validation of this model was confirmed by two sets of experimental data. Based on this model, three common types of middle ear malformations, i. e. incudostapedial joint defect, incus fixation and malleus fixation, and stapes fixation, were simulated by changing the structure and material properties of the corresponding tissue. Then, the effect of these three common types of middle ear malformations on energy absorbance was investigated by comparing the corresponding energy absorbance. The results showed that the incudostapedial joint defect significantly increased the energy absorbance near 1 000 Hz. The incus fixation and malleus fixation dramatically reduced the energy absorbance in the low frequency, which made the energy absorbance less than 10% at frequencies lower than 1 000 Hz. At the same time, the peak of energy absorbance shifted to the higher frequency. These two kinds of middle ear malformations had obvious characteristics in the wideband acoustic immittance test. In contrast, the stapes fixation only reduced the energy absorbance in the low frequency and increased energy absorbance in the middle frequency slightly, which had no obvious characteristic in the wideband acoustic immittance test. These results provide a theoretical reference for the wideband acoustic immittance diagnosis of middle ear malformations in clinic.

A requirement for Fgfr2 in middle ear development.

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Auditory ossicles: a potential biomarker for maternal and infant health in utero.

Background: Carbon (δ13C) and nitrogen (δ15N) isotope ratios of collagen from teeth and bone are used to study human nutrition and health. As bones are constantly remodelling throughout life, isotopic values of bone collagen represent an average of several years. In contrast, human teeth do not remodel and their primary dentine contains only the isotopic data from the time of formation. In contrast to all other bones, human auditory ossicles also appear not to remodel. As they develop in utero and finish formation in the first 2 years of life, their collagen should also represent isotopic values of these two relatively short periods.Aim: By comparing δ13C and δ15N data from ossicles and incremental dentine, this study aims to investigate how two developmental periods of the ossicles, in utero and the first 2 years of life, reflect in collagen obtained from the ossicles.Subject and methods: Ossicle and tooth samples of 12 individuals aged 0.5 ± 0.4 years to 13 ± 1 years from the nineteenth century St. Peter's burial ground in Blackburn were collected and processed to obtain bulk bone and incremental dentine collagen which was measured for δ13C and δ15N.Results: Averaged δ13C and δ15N of ossicles are lower when compared to every age group except after 3 years of age. Average offset between ossicles and dentine of different groups ranges from 0.4-0.9‰ for δ13C and from 0.3-0.9‰ for δ15N, with highest counterbalance at birth and after the first 5 months after birth.Conclusions: There appears to be a systematic offset between the dentine and ossicle data. It seems that the second phase of development does not influence the isotopic values of collagen significantly and the data we are obtaining from ossicles represents the in utero period.

Peripheral Hearing Structures in Fishes: Diversity and Sensitivity of Catfishes and Cichlids.

Fishes have evolved an astonishing diversity of peripheral (accessory/ancillary) auditory structures to improve hearing based on their ability to transmit oscillations of gas bladder walls to the inner ears. So far it is unclear to what degree the size of the bladder and the linkage to the ear affect hearing in fishes. An interfamilial study in catfishes revealed that families which possess large, single swim bladders and one to four Weberian ossicles were more sensitive at higher frequencies (≥1 kHz) than families which have small, paired, and encapsulated bladders and one to two ossicles. An intrafamilial investigation in thorny catfishes (family Doradidae) revealed that small differences in bladder morphology did not affect hearing similarly. Members of the cichlid family possess an even larger variation in peripheral auditory structures than catfishes. The linkage between the swim bladder and ear can either be present via anterior extensions of the bladder or be completely absent (in contrast to catfishes). Representatives having large bladders with extensions had the best sensitivities. Cichlids lacking extensions had lower sensitivities above 0.3 kHz. Species with a vestigial swim bladder exhibited a smaller hearing bandwidth than those with larger swim bladder (maximum frequency: 0.7 kHz vs. 3 kHz). Catfishes and cichlids reveal that larger gas bladders and more pronounced connections between the swim bladder and the inner ear result in improved hearing at higher frequencies. The lack of a connection between a large bladder and the inner ear does not necessarily result in a smaller detectable frequency range.

The architecture of the middle ear in the small Indian mongoose (Herpestes Javanicus).

The small Indian mongoose (Herpestes javanicus) is native to the Middle East, Iran and much of southern Asia. For this study the middle ears of a total of 6 adult small Indian mongooses, both fresh and museum samples were explored by using of dissection and plain radiography. On the one hand, at least in some species of the mongoose vocalisations and hearings play a critical role in coordinating behaviours. On the other hand, the ear region has provided useful character relevant for mammalian phylogeny. So, the aim of the present study is a brief discussion of the various anatomic particularities of the middle ear based on a combination of existing data and the results of the authors' study in the small Indian mongoose.

Anatomic and morphometric examination of auditory ossicles in sheep.

It is important to know the morphometry of the auditory ossicles for middle ear surgical applications. The present study aims to investigate the morphometric measurements of sheep auditory ossicles and the relationship between these ossicles. In this study, 100 malleus, incus and stapes of 50 Akkaraman sheep were examined using at trinocular stereo microscope and a total of 19 morphometric measurements were taken. The mean values of the morphometric measurements were obtained, and correlation analysis was performed between each part of each auditory ossicle. Similarities were found between the measurements of the lengths of the malleus, manubrium mallei and incus, and the width of the base of the stapes with morphometric measurements in human auditory ossicles. A significant positive correlation was found between the length of the malleus with the length of the manubrium mallei, between the length of the incus with the lengths of the long crus and corpus incudis, between the length of the stapes with the lengths of the rostral crus, caudal crus, caput stapedis, and the length and width of the intercrural foramen. Due to the anatomic similarities between sheep and human auditory ossicles, it was concluded that the auditory ossicles of sheep are suitable for use in the training of human ear surgery applications.

Middle Ear Ossicular Joint Changes in Type 2 Diabetes Mellitus: A Histopathological Study.

OBJECTIVES: Although previous research has indicated inner ear changes in diabetes mellitus (DM) patients, no prior study has explored the middle ear, particularly the ossicles and their joints, in DM patients. This study aimed to investigate whether type 2 DM is associated with middle ear changes, specifically affecting the ossicular chain and joints. METHODS: This study included 47 ears from 25 patients with DM (male = 13, female = 12, age: 51.0 ± 20.5) and age- and sex-matched controls (male = 10, female = 10, age: 54.8 ± 15.9) (sex; p = 1.000, Age; p = 0.991). Otopathological evaluations of the auditory ossicles and incudomalleolar joint (IMJ) were performed using light microscopy. RESULTS: In the IMJ of DM cases, malleus hyalinized cartilage (Malleus hC) and incus hyalinized cartilage (Incus hC) were significantly increased compared with control cases (Malleus hC; DM, 34.17 ± 9.71 µm vs. control 21.96 ± 4.16 µm, p < 0.001) (Incus hC; DM 35.11 ± 10.12 µm vs. control 22.42 ± 4.368 µm, p < 0.001). In addition, bone-line distance was significantly longer than in DM cases than control cases (DM 266.72 ± 59.11 µm vs. control 239.81 ± 35.56 µm p = 0.040). On the other hand, joint discus distance was longer in the control group than in DM cases (DM 96.84 ± 36.80 µm vs. Control 113.63 ± 23.81 µm, p = 0.001). CONCLUSIONS: This study reveals a notable increase in the hyalinized cartilage layer and bone-line distance accompanied by reducing joint discus distance within the IMJ in DM cases. These findings suggest that DM may influence microjoints, such as the IMJ, and potentially impact auditory function. EVIDENCE LEVEL: N/A Laryngoscope, 134:2871-2878, 2024.

Ontogeny of the malleus in Mesocricetus auratus (Mammalia, Rodentia): Systematic and functional implications for the muroid middle ear.

The three mammalian auditory ossicles enhance sound transmission from the tympanic membrane to the inner ear. The anterior anchoring of the malleus is one of the key characters for functional classification of the auditory ossicles. Previous studies revealed a medial outgrowth of the mallear anterior process, the processus internus praearticularis, which serves as an anchor for the auditory ossicle chain but has been often missed due to its delicate nature. Here we describe the development and morphology of the malleus and its processus internus praearticularis in the cricetine rodent Mesocricetus auratus, compared to selected muroid species (Cricetus cricetus, Peromyscus maniculatus, and Mus musculus). Early postnatal stages of Mesocricetus show the formation of the malleus by fusion of the prearticular and mallear main body. The processus internus praearticularis forms an increasing broad lamina fused anteriorly to the ectotympanic in adult stages of all studied species. Peromyscus and Mus show a distinct orbicular apophysis that increases inertia of the malleus and therefore these species represent the microtype of auditory ossicles. In contrast, the center of mass of the malleus in the studied Cricetinae is close to the anatomical axis of rotation and their auditory ossicles represent the transitional type. The microtype belongs to the grundplan of Muroidea and is plesiomorphic for Cricetidae, whereas the transitional type evolved several times within Muroidea and represents an apomorphic feature of Cricetinae.

Anatomical, Histological, and Morphometrical Investigations of the Auditory Ossicles in Chlorocebus aethiops sabaeus from Saint Kitts Island.

Otological studies rely on a lot of data drawn from animal studies. A lot of pathological or evolutionary questions may find answers in studies on primates, providing insights into the morphological, pathological, and physiological aspects of systematic biological studies. Our study on auditory ossicles moves from a pure morphological (macroscopic and microscopic) investigation of auditory ossicles to the morphometrical evaluation of several individuals as well as to some interpretative data regarding some functional aspects drawn from these investigations. Particularities from this perspective blend with metric data and point toward comparative elements that might also serve as an important reference in further morphologic and comparative studies.

Prevention of Hypomineralization In Auditory Ossicles of Vitamin D Receptor (Vdr) Deficient Mice.

Intact mineralization of the auditory ossicles - the smallest bones in the body - is essential for sound transmission in the middle ear, while ossicular hypomineralization is associated with conductive hearing loss. Here, we performed a high-resolution analysis of the ossicles in vitamin D receptor deficient mice (Vdr-/- ), which are characterized by hypocalcemia and skeletal mineralization defects, and investigated whether local hypomineralization can be prevented by feeding a calcium-rich rescue diet (Vdr-/- res ). In Vdr-/- mice fed a regular diet (Vdr-/- reg ), quantitative backscattered electron imaging (qBEI) revealed an increased void volume (porosity, p<0.0001) along with lower mean calcium content (CaMean, p=0.0008) and higher heterogeneity of mineralization (CaWidth, p=0.003) compared to WT mice. Furthermore, a higher osteoid volume per bone volume (OV/BV; p=0.0002) and a higher osteocyte lacunar area (Lc.Ar; p=0.01) were found in histomorphometric analysis in Vdr-/- reg mice. In Vdr-/- res mice, full rescue of OV/BV and Lc.Ar (both p>0.05 vs. WT) and partial rescue of porosity and CaWidth (p=0.02 and p=0.04 vs. WT) were observed. Compared with Hyp mice, a model of X-linked hypophosphatemic rickets, Vdr-/- reg mice showed a lower osteoid volume in the ossicles (p=0.0002), but similar values in the lumbar spine. These results are consistent with later postnatal impairment of mineral homeostasis in Vdr-/- mice than in Hyp mice, underscoring the importance of intact mineral homeostasis for ossicle mineralization during development. In conclusion, we revealed a distinct phenotype of hypomineralization in the auditory ossicles of Vdr-/- mice that can be partially prevented by a rescue diet. Since a positive effect of a calcium-rich diet on ossicular mineralization was demonstrated, our results open new treatment strategies for conductive hearing loss. Future studies should investigate the impact of improved ossicular mineralization on hearing function.

Application value of a deep learning method based on a 3D V-Net convolutional neural network in the recognition and segmentation of the auditory ossicles.

Objective: To explore the feasibility of a deep learning three-dimensional (3D) V-Net convolutional neural network to construct high-resolution computed tomography (HRCT)-based auditory ossicle structure recognition and segmentation models. Methods: The temporal bone HRCT images of 158 patients were collected retrospectively, and the malleus, incus, and stapes were manually segmented. The 3D V-Net and U-Net convolutional neural networks were selected as the deep learning methods for segmenting the auditory ossicles. The temporal bone images were randomized into a training set (126 cases), a test set (16 cases), and a validation set (16 cases). Taking the results of manual segmentation as a control, the segmentation results of each model were compared. Results: The Dice similarity coefficients (DSCs) of the malleus, incus, and stapes, which were automatically segmented with a 3D V-Net convolutional neural network and manually segmented from the HRCT images, were 0.920 ± 0.014, 0.925 ± 0.014, and 0.835 ± 0.035, respectively. The average surface distance (ASD) was 0.257 ± 0.054, 0.236 ± 0.047, and 0.258 ± 0.077, respectively. The Hausdorff distance (HD) 95 was 1.016 ± 0.080, 1.000 ± 0.000, and 1.027 ± 0.102, respectively. The DSCs of the malleus, incus, and stapes, which were automatically segmented using the 3D U-Net convolutional neural network and manually segmented from the HRCT images, were 0.876 ± 0.025, 0.889 ± 0.023, and 0.758 ± 0.044, respectively. The ASD was 0.439 ± 0.208, 0.361 ± 0.077, and 0.433 ± 0.108, respectively. The HD 95 was 1.361 ± 0.872, 1.174 ± 0.350, and 1.455 ± 0.618, respectively. As these results demonstrated, there was a statistically significant difference between the two groups (P < 0.001). Conclusion: The 3D V-Net convolutional neural network yielded automatic recognition and segmentation of the auditory ossicles and produced similar accuracy to manual segmentation results.

CCDC154 Mutant Caused Abnormal Remodeling of the Otic Capsule and Hearing Loss in Mice.

Osteopetrosis is a rare inherited bone disease characterized by dysfunction of osteoclasts, causing impaired bone resorption and remodeling, which ultimately leads to increased bone mass and density. Hearing loss is one of the most common complications of osteopetrosis. However, the etiology and pathogenesis of auditory damage still need to be explored. In this study, we found that a spontaneous mutation of coiled-coil domain-containing 154 (CCDC154) gene, a new osteopetrosis-related gene, induced congenital deafness in mice. Homozygous mutant mice showed moderate to severe hearing loss, while heterozygous or wild-type (WT) littermates displayed normal hearing. Pathological observation showed that abnormal bony remodeling of the otic capsule, characterized by increased vascularization and multiple cavitary lesions, was found in homozygous mutant mice. Normal structure of the organ of Corti and no substantial hair cell or spiral ganglion neuron loss was observed in homozygous mutant mice. Our results indicate that mutation of the osteopetrosis-related gene CCDC154 can induce syndromic hereditary deafness in mice. Bony remodeling disorders of the auditory ossicles and otic capsule are involved in the hearing loss caused by CDCC154 mutation.

Evaluation of the middle ear in water buffaloes (Bubalus bubalis) by gross anatomy and cone-beam computed tomography.

BACKGROUND: The purpose of this study was to provide a description of gross middle ear morphology in water buffaloes, augmented with additional data on the osseous structures of middle ear derived from cone-beam computed tomography (CBCT). MATERIALS AND METHODS: Skulls of 10 young adult male water buffaloes were used to examine their middle ears. RESULTS: Anatomical features noted included the presence of tympanic cells in the tympanic bulla, the location of malleus head and neck, and all of incus in the dorsal epitympanic recess, the oval tympanic membrane, absence of a prominent notch on the articular surface of malleus, positional variations of the lateral process of malleus relative to the muscular process and muscular process relative to the rostral process of malleus, absence of complete coverage of the articular facet of malleus head by incus body, and presence of the lenticular process of incus. In CBCT images, the osseous part of external acoustic meatus, the petrous part of temporal bone and the details of the ossicles were seen, except for stapes. CONCLUSIONS: Although tympanic membrane, malleus and stapes of water buffaloes are similar to those of ox, the incus of water buffaloes is more similar to that of goats. The heaviest ossicles among the ruminants studied belonged to water buffaloes; the mean length of malleus head and neck, total length and width of incus body as well as length of stapes head were greatest in water buffaloes too. The auditory ossicles of water buffaloes show 'transitional type' morphological characteristics. These features suggest a relatively wide frequency range of hearing, but not one biased towards especially low or especially high frequencies.

Morphological and morphometrical study of the auditory ossicles in chinchilla.

This study is meant to illustrate and describe the features of the auditory ossicles of the chinchilla (Chinchilla lanigera), one of the species used more and more frequently in otology and ear surgery as animal model. Cephalic extremities of 12 C. lanigera individuals obtained from a private farm, where this species was bred for fur, were used in this study. The ossicles were obtained either by direct surgical harvesting by mastoid approach or after a dermestid beetle exposure followed by anatomical dissection. The three ossicles that form the assembly are the malleus, incus and stapes. After the removal of these ossicles, a series of anatomical descriptions were made, followed by seriate sets of measurements. The malleus and incus form a joined-single unit called the maleo-incal complex, with an elongated straight appearance, also due to the development of the anterior process. The handle of the malleus and the long process of incus are almost perpendicular to the main axis of the maleo-incal complex. The presence of the muscular process on the handle of the malleus is recorded. The overall shape of the incus is given by the uneven development of the two processes and the reduced neck part. The stapes is the smallest of the components that maintains the well-known architecture in accordance with the general model. The morphology of all three ossicles is backed by a series of measurements, some standard, some adapted to the morphology of the ossicles. From the very reduced comparative metrical data at our disposal, our study presents an average of 10% lower values for the ones presented earlier by other researchers in the same species.

The vertebrate middle and inner ear: A short overview.

The evolution of the various hearing adaptations is connected to major structural changes in nearly all groups of vertebrates. Besides hearing, the detection of acceleration and orientation in space are key functions of this mechanosensory system. The symposium "show me your ear - the inner and middle ear in vertebrates" held at the 11th International Congress of Vertebrate Morphology (ICVM) 2016 in Washington, DC (USA) intended to present current research addressing adaptation and evolution of the vertebrate otic region, auditory ossicles, vestibular system, and hearing physiology. The symposium aimed at an audience with interest in hearing research focusing on morphological, functional, and comparative studies. The presented talks and posters lead to the contributions of this virtual issue highlighting recent advances in the vertebrate balance and hearing system. This article serves as an introduction to the virtual issue contributions and intends to give a short overview of research papers focusing on vertebrate labyrinth and middle ear related structures in past and recent years.

Synchrotron radiation imaging revealing the sub-micron structure of the auditory ossicles.

PURPOSE: Synchrotron-based X-ray Phase Contrast Imaging (SR X-PCI) allows, thanks to a highly coherent and powerful X-ray beam, the imaging of surface and cross-sectional tissue properties with high absorption-contrast. The objective of this study is to investigate the sub-micron structure of the ossicular chain. The understanding of its morphological properties at sub-micron scale will help to refine the understanding of its structural properties. The investigation of intact, non-decalcified and unstained ossicular bones allows to study the spatial relationship between surface properties, internal structure and tomographical slides. MAIN RESULTS: The tomography datasets with a pixel size of 0.65 µm were reconstructed and 3D volume rendering models of all specimens were analyzed. Based on surface models, the surfaces of the articulations, the insertion of the tensor tympani and stapedial muscle tendons and the nutritional foramina, where the vessels penetrate the ossicles, were visualized. Moreover, a branched network of inner channels could be represented and its connection to the nutritional foramen was demonstrated. Looking at the tomographic structure of the three ossicles a mineralization pattern for every auditory bone was described, indicating a considerable variation throughout the bones. CONCLUSIONS: This study investigates the submicron-structure of the auditory ossicles at a pixel size of 0.65 µm, which is to the best of our knowledge the highest resolution reported in the investigation of the human auditory system so far. The provided data helps in the further understanding of the anatomical conformation of the ossicular chain.

Development of the Human Incus With Special Reference to the Detachment From the Chondrocranium to be Transferred into the Middle Ear.

The mammalian middle ear represents one of the most fundamental features defining this class of vertebrates. However, the origin and the developmental process of the incus in the human remains controversial. The present study seeks to demonstrate all the steps of development and integration of the incus within the middle ear. We examined histological sections of 55 human embryos and fetuses at 6 to 13 weeks of development. At 6 weeks of development (16 Carnegie Stage), the incus anlage was found at the cranial end of the first pharyngeal arch. At this stage, each of the three anlagen of the ossicles in the middle ear were independent in different locations. At Carnegie Stage 17 a homogeneous interzone clearly defined the incus and malleus anlagen. The cranial end of the incus was located very close to the otic capsule. At 7 and 8 weeks was characterized by the short limb of the incus connecting with the otic capsule. At 9 weeks was characterized by an initial disconnection of the incus from the otic capsule. At 13 weeks, a cavity appeared between the otic capsule and incus. Our results provide significant evidence that the human incus developed from the first pharyngeal arch but independently from Meckel's cartilage. Also, during development, the incus was connected with the otic capsule, and then it was detached definitively. The development of the incus in humans provides evidence that this ossicle is homologous to the quadrate. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.