Developmental Disruptions of the Human Stapes

Abstract

Objective:

To evaluate and classify developmental malformations of the human stapes.

Methods:

25 temporal bone specimens from 18 patients with congenital stapes malformations were identified in the Mass Eye and Ear temporal bone collection. Serial sections stained with Hematoxylin and Eosin were examined by light microscopy and the morphology of the stapes was compared to age matched controls.

Results:

Each case of stapes malformation could be classified into one of four malformation types based on our current understanding of the embryologic origin of the subunits of the stapes and timing of development. 27% of stapes malformations had a Type I morphology characterized by a hypoplastic or absent inner footplate and hypoplastic to absent mesoderm footplate or oval window. The crura and capitulum may be absent, monopodal or dysmorphic. 11% expressed a Type II malformation with dysmorphic or monopodal capitulum and crura and a fixed footplate. 27% were of Type III with a dysmorphic or monopodal capitulum and or crura. The footplate, and thereby oval window is present and without fixation. The most common malformation, Type IV, was isolated footplate fixation observed in 33% of cases.

Conclusions:

Malformations of the human stapes follow consistent patterns of early or late disruptions of the stapes subunits of mesodermal and/or neural crest origin. While the molecular events, including temporal coordination, that lead to a normally formed stapes are not yet fully understood, the observed patterns of human stapes malformation can be consistently classified into one of four patterns of developmental disruption.

INTRODUCTION

Conductive hearing loss is one of the most common forms of human hearing loss. Conductive hearing loss can be the result of congenital hearing bone (ossicle) malformations that are estimated to be present in 1 per 15,000 births [1]. The human ossicles are the result of long evolutionary processes that transformed structural jaw elements to auditory elements in terrestrial vertebrates [2]. Our understanding of the coordinated molecular regulation of ossicle formation is incomplete. Recent studies [2–9] have begun to better inform our understanding of the development of the ossicles including the third bone of hearing the stapes, which is the focus of the present study. These studies point to a dual neural crest and mesoderm origin of the stapes.

In this study we evaluated and classified human temporal bone specimens from a cohort of patients with syndromic and non-syndromic developmental defects of the stapes. We propose a new classification system of developmental stapes malformations that is informed by our understanding of the dual origin and temporal events regulating stapes development. This categorization of stapes anomalies is informative to the clinician’s decision making in the treatment of congenital conductive hearing loss. Namely abnormalities in the mesoderm derived subunits of the stapes point to more severe dysmorphologies and would steer practitioners to more conservative rehabilitation options.

MATERIAL AND METHODS

Temporal bones with congenital ossicular anomalies were identified in the Massachusetts Eye and Ear temporal bone collection. The clinical history was collected during life through enrollment in the National Institute on Deafness and Communication Disorders (NIDCD) National Temporal Bone, Hearing, and Balance Pathology Resource Registry.

Temporal bones were harvested and prepared for light microscopy by fixation in formalin followed by standard processing for histologic examination, including decalcification with ethylenediamine tetra-acetic acid (EDTA) and celloidin embedding. Specimens were sectioned serially in the horizontal plane at a thickness of 20 μm. Every tenth section was then stained with Hematoxylin and Eosin and mounted on a glass slide. The slides were examined by light microscopy and compared to age matched controls.

The subunit of each stapes including the capitulum, crura, footplate, annular ligament and annular ligament was categorized as absent, dysmorphic or normal by both authors. The malformations were then grouped into four categories based on our current understanding of the embryologic origin of the subunits of the stapes and timing of development. The capitulum, crura, and inner footplate are of neural crest origin. The outer footplate, annulus, and oval window are of mesodermal origin [5,9].

All specimens in the Type I category have disruptions in the neural crest derived subunits presumed to occur early enough in development to also disrupt the morphology of the mesoderm derived oval window. Type II malformations have disruptions of the neural crest derived subunits and more restricted malformations of the mesoderm derived subunits with the presence of an oval window. Type III stapes anomalies have malformations attributed to the later endochondral ossification of the neural crest derived subunits of the stapes. Type IV malformations are restricted to isolated stapes footplate fixation. The authors also independently described the morphology of the incus, malleus, middle ear, facial nerve and cochlea for each case as normal or abnormal compared to age matched controls.

RESULTS

Cases

25 temporal bone specimens from 18 patients with congenital stapes malformations were identified (bilateral malformations, n= 7; unilateral malformation, n = 11). Thirteen of these donors (72.22%) had known syndromic diseases. We excluded cases of otosclerosis and osteogenesis imperfecta. The demographics of the 18 donors are listed in Table 1.

Table 1.

Patient demographics.

Type	Age in years	Gender	Syndrome	Hearing Loss	Other Anomalies
I	30	M	No	Unknown
I	>1	F	Nager	Unknown	Ma,In,Me
I	>1	F	CHARGE	Unknown	Co
I	>1	F	DiGeorge	Unknown	Ma,In,FN,STM,Co
I	>1	M	Trisomy 22	Unknown	Ma,In,STM,Co
II	2	M	Otopalatodigital	Conductive	Ma,In
II	>1	F	No	Unknown	In,Me,Co
III	3	F	Neural crestopathy	Sensorineural	Ma,In,Co
III	18	M	Kleefstra	Unknown	Co
III	>1	F	Treacher Collins	Unknown
III	>1	F	Kleeblattschadel	Unknown	Ma,In
III	>1	M	Trisomy 13–15 (D/D translocation)	Unknown	Ma,In,FN,Co
IV	47	M	Spinal dysraphism and tethered cord	Mixed loss	Co
IV	56	F	No	Mixed Loss
IV	>1	M	Nager	Unknown	Ma,In,Me
IV	>1	M	No	Unknown
IV	19	M	No	Mixed loss	Ma,In
IV	78	M	NOG-SSD	Mixed

Ma=malleus, In-incus, Co=cochlea, Me=middle ear,FN=facial nerve, STM=stapedius muscle.

Stapes Malformations

The malformations of the stapes were categorized by the morphology of the capitulum, crura, footplate, annular ligament and oval window for each case as listed in Table 2. Among the seven donors with bilateral malformations, all had similar stapes malformations bilaterally, and only one donor had an asymmetric malformation of the malleus and incus. The cases are therefore listed by donor in Table 2 rather than by individual temporal bone.

Table 2.

Morphology of Stapes. The morphology of each stapes subunit is described and cases are listed by type according to the proposed classification system.

Type	Capitulum	Crura	Footplate	Annular Ligament	Oval Window
I	absent	absent	absent	absent	absent
I	absent	absent	absent	absent	absent
I	monopodal	monopodal	absent	absent	absent
I	dysmorphic	dysmorphic	absent	absent	absent
I	dysmorphic	dysmorphic	hypoplastic	dysmorphic	hypoplastic
II	dysmorphic	dysmorphic	normal	fixation	present
II	monopodal	monopodal	normal	fixation	present
III	absent	absent	dysmorphic	present	present
III	monopodal	monopodal	dysmorphic	present	present
III	dysmorphic	dysmorphic	dysmorphic	present	present
III	normal	dysmorphic	normal	present	present
III	normal	dysmorphic	normal	present	present
IV	normal	normal	normal	fixation	normal
IV	normal	normal	normal	fixation	normal
IV	normal	normal	normal	fixation	normal
IV	normal	normal	normal	fixation	normal
IV	normal	normal	normal	fixation	normal
IV	normal	normal	normal	fixation	normal

Green=normal subunit morphology,yellow= abnormal subunit morphology, red=absent subunit.

Capitulum of the Stapes

Seven cases had a dysmorphic capitulum. Of these seven, three were associated with a monopodal stapes (Figure 1). Of those with a monopodal stapes one carried a diagnosis of Treacher Collins syndrome and two had no definitive syndromic diagnosis but died within the first year of life secondary to systemic developmental anomalies.

Figure 1.

Monopodal stapes (asterix). The capitulum and crurae of the stapes are of neural crest cell origin. Monopodal stapes are observed in Type I, II, and III malformations.

Three cases had an absent capitulum. Of these three, all were missing crurae and only one had a footplate and oval window.

Crurae of the Stapes

Ten cases had dysmorphic crurae including three with a monopodal structure. Three cases with dysmorphic crurae, but normal capitulum, had thickened crurae suggestive of incomplete endochondral ossification (Figure 4–C). None of the identified cases had a persistent stapedial artery.

Figure 4.

a-d. Representative sections of each malformation by type. a. Type I There is no footplate or oval window. The crura and capitulum are monopodal (asterix) b. Type II Dysmorphic capitulum and crura (arrowheads) and a fixed footplate (arrow). The the oval window is present. c.Type III The footplate and oval window are present and without fixation. The stapes crura and capitulum are thick and dysmorphic. d. Type IV : Isolated fixation of the stapes footplate (arrow).

Three cases had absent crurae. Two of these had no footplate and no oval window.

Footplate

The footplate was dysmorphic in five cases of which one was hypoplastic (Figure 2).

Figure 2.

A hypoplastic footplate (arrow heads). The stapes footplate is of neural crest and mesodermal origin. FN=Facial nerve.

There were four missing footplates, one associated with a monopodal superstructure, one with a dysmorphic structure, and two with no superstructure.

Annular ligament

There were eight cases of fixation of the footplate. Five of these were isolated anomalies with otherwise normal stapes development (Figure 3 and 4–D).

Figure 3.

Congenital fixation of the footplate (arrow) is the most commonly observed stapes malformation.

Four cases had a missing annular ligament, all with missing footplates.

Oval Window

There were four cases with absent oval windows all with absent footplate and annular ligament.

One case had a hypoplastic oval window associated with a hypoplastic footplate but present annular ligament.

Malleus and Incus Malformation

Ten cases had concurrent malleus and incus malformations. Of those ten, eight had known syndromic disease including Neural Crestopathy, Nager syndrome, Otopalatodigital syndrome, Kleeblattschadel syndrome, Di George syndrome, trisomy 22, and trisomy 13–15.

Facial Nerve

Only one case (with Di George syndrome) exhibited a hypoplastic facial nerve with an abnormal course. The facial nerve consisted of the intracanalicular component, geniculate ganglion and greater superficial petrosal nerve. The intratemporal motor division was absent.

Classification

Each case could be classified into one of four malformation types based on our current understanding of the embryologic origin of the subunits of the stapes and timing of development. Figure 4 shows a representative example of each malformation by type.

Type I:

Stapes malformations with a hypoplastic or absent inner footplate and hypoplastic to absent mesoderm footplate or oval window. The crura and capitulum may be absent, monopodal or dysmorphic. This is consistent with early disruption of neural crest cell derived stapes subunits which in turn affects the development of the mesoderm derived outer footplate, annular ligament and oval window. This type of malformation was present in five of 18 cases.

Type II:

Stapes malformations with dysmorphic or monopodal capitulum and crura and a fixed footplate. The footplate and oval window are otherwise developed. This points to more restricted developmental anomalies of the neural crest and mesodermal derived subunits. Two of eighteen cases exhibited this type of malformation.

Type III:

Stapes with dysmorphic or monopodal capitulm and or crura. The footplate, and thereby oval window is present and without fixation. These are anomalies affecting the endochondral ossification of the neural crest derived portion of the stapes including capitulum, crura and inner footplate. Five of 18 cases had this type of malformation.

Type IV:

Isolated footplate fixation. This is an isolated anomaly of the mesodermal derived annular ligament. This was the most common malformation occurring in six of 18 cases.

DISCUSSION

The stapes is the smallest bone in the human body. It is stirrup shaped and conducts vibration of sound from the incus to the inner ear. It is composed of a head or capitulum which articulates with the incus through the incudo-stapedial joint, and two limbs or crura that connect to a footplate. The footplate is the base of the stapes that sits in the oval window connected to the otic capsule by an annular ligament. The annular ligament allows vibration of the footplate thereby transfering sound to the scala vestibuli. Congenital malformations of the stapes can result in conductive hearing loss.

Studies involving comparative anatomy, embryology and paleontology have suggested that the primary jaw articulation, along with the hyomandibula (columella in chick and reptiles) were incorporated into the middle ear to form a three-ossicle chain [2]. The stapes is the mammalian equivalent of the single non-mammalian terrestrial vertebrate columella derived from the rostral end of Reichert’s cartilage [4]. When disrupted during embryological development, the recapitualtion of these evolutionary events can manifest in syndromes where facial skeletal malformations occur with ossicular anomalies. Our case series included multiple examples of syndromic facial skeletal and ossicular malformations including Nager, Treacher Collins, Otopalatodigital, CHARGE, and Kleefstra syndrome.

Congenital middle ear malformations can be part of a complex syndrome or isolated ear disorders. In our cohort 72.2% of donors with stapes malformation had other malformations associated with syndromes. This is higher than reported in other developmental human studies where up to 30% of ear malformations are associated with defined syndromes [10,11,12]. This discrepancy is explained by the inherent selection bias of a Registry, our study selection of looking at the stapes only, and by the fact that our cohort does not include more restricted ear anomalies occuring later in development e.g. malformations of the pinna.

The head, crura and inner footplate of the stapes are of neural crest cell origin from the second pharyngeal arch [4,5]. The outer part of the footplate attaches to the otic capsule via the annular ligament. The outer footplate, annular ligament and surrounding otic capsule are of mesodermal origin. This points to a dual embryological origin of the stapes: neural crest and mesoderm [5,9].

The development of the mesoderm derived oval window including the annular ligament and otic capsule is believed to occur temporally with, but likely not dependent upon, neural crest footplate development. This interpretation is based on the observation that mutant mice lacking a neural crest stapes can develop a small oval window [13]. We identifed 5 cases, classified as Type I, with a hypoplastic or absent inner footplate and hypoplastic to absent mesoderm footplate or oval window. In all five cases the crura and capitulum were affected being absent, monopodal or dysmorphic. This is consistent with early disruption of neural crest cell derived stapes subunits which in turn affect the development of the mesoderm derived outer footplate, annular ligament, and oval window. Notably four of the five cases died in the neonatal period suggestive of more early global disruption of embryological development.

Two of the eighteen cases exhibited more restricted abnormalities of the stapes subunits of neural crest and mesoderm origin. These were classified separately as type II suggesting disruption of events occuring later in development than seen in type I. As our understanding of the molecular regulation of neural crest and mesoderm subunits of the stapes after footplate and oval window development improves, this classification can be further refined.

Human temporal bone studies show that in the 8 week fetus the superstructure (head and crura) are in the shape of an annulet, and by the 10th week have taken a stirrup shape. The shape continues to differentate into the 7th month [14]. Five of the eighteen cases showed anomalies associated with this process of endochondral ossification of the neural crest cell origin subunits of the stapes and were classified as type III anomalies.

The most common malformation of the stapes we identified was isolated fixation of stapes footplate occuring in 6 of 18 cases. This isolated fixation, type IV stapes malformation, is presumed to be the consequence of a late mesodermal event. Fixation of the footplate was not unique to this type however. Stapes fixation was also observed in conjuction with other anomalies of the stapes, namely type II malformations. One of the six cases of isolated stapes fixation had an autosomal dominant NOG-related symphalangism disorder (NOG-SSD). The NOG gene encodes the noggin protein which is a known bone morphogenetic protein antagonist critical for normal skeletal and joint development [15]. As there was a mix of syndromic and non sydromic patients with this type IV isolated stapes fixation it is expected that molecular dysregulations other than noggin expression can also express this phenotype.

Formation of facial nerve begins during the fourth week of gestation. The formation of the facial nerve is reported to be intimately related to the development of the stapes, the annular ligament, the oval window, and the vestibular component of the otic capsule [16,17]. There are a few reports however that describe abnormal developmental relationships between facial nerve and stapes development. We only found one donor (5.5%) with a hypoplastic facial nerve suggesting unique molecular, if not temporal, regulation.

Histopathologic examination showed that 3 donors (5 temporal bones) with stapes malformation also exhibited incus and malleus malformation; 1 donor (2 temporal bones) with fixation of footplate and incus and malleus malformation; 4 donors (6 temporal bones) with stapes malformation, fixation of footplate and incus and malleus malformation (2 with Nager syndrome; 1 with Otopalatodigital syndrome). The malleus and incus form from Meckel’s cartilage. Ossification of the incus begins at 16

Weeks of gestation followed by the malleus between gestation weeks 16 and 17 [4]. There was no consistent pattern of malleus and/or incus malformation to stapes malformation indicative of the molecular and temporal heterogeneity in individual ossicular development.

CONCLUSIONS

Malformations of the human stapes follow consistent patterns of early or late disruptions of the stapes subunits of mesodermal and/or neural crest origin. This categorization can can serve to inform radiographic interpretations of stapes morphology. Recognition of anomalies affecting both the neural crest and mesoderm derived subunits point to more global disruptions and in turn isolated footplate fixations point to a more restricted process that may be amenable to surgical correction. While the molecular events, including temporal coordination, that lead to a normally formed stapes are not yet fully understood, the observed patterns of human stapes malformation can be consistently classified into one of four patterns of developmental disruption. In the first pattern, an early disruption of neural crest cell derived stapes subunits affects the development of the mesoderm derived outer footplate, annular ligament and oval window. In the second, there are more restricted developmental anomalies of the neural crest and mesodermal derived subunits. In this second group the capitulum and crura are dysmorphic or monopodal and while the footplate may be fixed, the footplate and oval window are otherwise developed. The third and fourth types of malformations exhibit a more restricted dysmorphology suggesting, albeit not conclusively, disruption of events occuring later in stapes development that seen in the first and second group. The third group consists of anomalies affecting the endochondral ossification of the neural crest derived portion of the stapes including capitulum, crura and inner footplate. Finally the fourth and most common category is isolated fixation of the stapes. This points to disruption of an event restricted to the mesodermal derived annular ligament.