Carnegie stage
Gestational week
Gestational day (s)
Fetal diameter (mm)
9
3
19–21
1.5–2.5
10
4
22–23
2–3.5
11
4
23–26
2.5–4.5
12
4
26–30
3–5
13
5
28–32
4–6
14
5
31–35
5–7
15
5
35–38
7–9
16
6
37–42
8–11
17
6
42–44
11–14
18
7
44–48
13–17
19
7
48–51
16–18
20
8
51–53
18–22
21
8
53–54
22–24
22
8
54–56
23–28
23
8
56–60
27–31
Comparison of embryonic laryngeal and vocal fold development in utero in murine and humans
Week | Human | Murine | Embryonic day (E) |
|---|---|---|---|
3 | Laryngeal formation begins | Laryngeal formation begins | 9.5 |
5 | Lateral walls of the primitive laryngopharynx are almost meeting medially | Initiation of the larynx and VF with apposition of the lateral walls of the primitive laryngopharynx | 10.5 |
5 | Primitive laryngopharynx is bilaterally compressed | Fusion of lateral wall of primitive laryngopharynx | 11.5 |
7 | Establishment of blood flow to the epithelial lamina begins | Establishment of blood flow to the epithelial lamina; laryngotracheal septum is visible | 13.5 |
8 | Cartilages are formed | Epithelial lamina continues to separate; cartilages are visible | 15.5 |
VF separation is completed | 18.5 | ||
10 | VF are differentiated | VF fully separated and firmly attached to the thyroid and arytenoids | P0/birth |
Summary of fetal developmental key features
Stage of embryonic development | Week of development | Key features in embryonic laryngeal and vocal fold development |
|---|---|---|
Embryonic period | 3 | First indication of laryngeal development, the median pharyngeal groove appears |
4 | Signs of the separation of the esophagus from the trachea are noted | |
5 | Arytenoid swellings present; superior laryngeal nerve is identified | |
6 | Recurrent laryngeal nerve is identified | |
7 | Posterior cricoarytenoid, interarytenoid, cricothyroid, and lateral cricoarytenoid are identified and innervated | |
8 | Fusion along the posterior cricoid lamina signals the end of chondrification; arytenoids contain muscular and vocal processes; thyroid lamina almost met medially to fuse | |
8 | Arytenoids develop their muscular and vocal processes | |
Fetal period | 10 | True vocal folds and vestibular folds differentiate |
14 | Random “flutter-like” movement at the level of the glottis present | |
15 | Inconsistent tongue thrusting and swallow | |
16 | Inconsistent tongue cupping | |
17 | Periodic glottic movement noted through ultrasound | |
18 | Inconsistent tongue protrusion | |
21 | Consistent tongue thrusting | |
22 | Consistent swallow present | |
28 | Consistent tongue cupping and tongue protrusion | |
29 | Complex swallow present |
The first indication of the larynx appears during week 3 (stage 9) via the median pharyngeal groove [2, 5, 6]. During week 4 (stages 10–12), there are three key changes. The first series occur during stage 10, when the median pharyngeal groove is noted to include the laryngotracheal sulcus with developing pulmonary primordium caudally and identification of the oropharyngeal membrane and second branchial arch is noted [2, 5–10]. The second branchial arch gives rise to the hyoid bone, esophagus, laryngotracheal ridge, trachea, bronchial buds, and thyroid diverticulum by approximately week 5 (stage 15) [8]. Also during week 4 (stage 11), a single layer of the epithelium develops in the laryngotracheal sulcus [11–13], signs of separation of the esophagus from the trachea are noted, and the tracheoesophageal septum is visualized [2, 6, 10, 11]. Lastly (stage 12), motor/sensory fibers of cranial nerves (CNs) XII, V, VII, VIII, IX/X, and XI appear in that order, and the superior ganglia of CN X are identified [11, 14].
By week 5 (stages 13–15), undifferentiated mesenchyme is noted to surround the primitive glottic slit, the lateral walls of the primitive laryngopharynx are almost meeting medially, and tracheoesophageal separation occurs [2, 15–17]. Also of note, arytenoid swellings begin to develop, and the third and fourth branchial arches, which will form future laryngeal cartilages and the ansa cervicalis, are seen with conclusion of development in week 6 (stage 17) (Kallius, 1897, as cited by Zaw-Tun & Burdi) [8, 13, 17]. Week 5 is also significant for the beginning stages of development of the cricoid and arytenoid [2, 11, 16, 18] with the right and left arytenoid swellings aiding in identification of the embryonic laryngeal inlet (stage 14) [6] and full detachment of the trachea (Streeter, 1942 as cited by O’Rahilly & Boyden) [5, 7, 11]. The epithelial lamina of the larynx begins its formation (stage 14) [2, 6, 7] followed by increased cell density of the arytenoid swellings and identification of the wedge-shaped mesenchyme anteriorly and bilateral to the epithelial lamina that will give rise to the laryngeal musculature and cartilages (stage 15) [9, 11]. The superior laryngeal nerve (SLN) is also identifiable during this stage [2, 19].
During week 6 (stages 16–17), the epiglottal swelling is identifiable [13], the hyoid has begun to develop [2], and the cricoid begins its formation from laryngeal mesodermal anlage (stage 16) [13]. While some report that the arytenoids begin their development during stage 14, Crelin [8] reports them being identifiable during stage 16, and Zaw-Tun et al. [13] report that they are taking on a conical shape. Week 6 is also significant for the epithelial lamina consisting of two closely fused cell layers [20] and identification of the recurrent laryngeal nerve (RLN) [19].
During week 7 (stages 18–19), Lisser [21] noted that the thyroid and epiglottis have begun to develop (stage 18); however, research has also suggested that chondrification has initiated for the hyoid bone and thyroid lamina at this time [2, 6, 16]. The posterior cricoarytenoid (PCA), interarytenoid (IA), cricothyroid (CT), and lateral cricoarytenoid (LCA) muscles are identified [12, 16]. The cartilages are more adult-like in shape [12] with the epiglottis appearing concave (stage 19) [6]. Both the SLN and RLN are innervating their respective muscles with the CT, PCA, and IA being isolated, and the LCA is reliably differentiated from the thyroarytenoid (TA) [12, 16]. Also of note, the epiglottis is concave in shape, and the process of blood flow recanalization begins and is completed during week 8 (stage 23) with the glottis [6, 17].
Week 8 (stages 20–23) is significant for fusion along the posterior cricoid lamina and signals the end of chondrification [14]. The PCA is noted to be in its adult position [16], and the arytenoids have developed their muscular and vocal processes [13]. Toward the end of week 8, the ventricle is formed [8], and all intrinsic muscles are clearly recognizable (stage 22) [12].
By the end of week 8, the last of the structural changes of the larynx occur. The hyoid bone parallels the anterior border of the thyroid cartilage [22, 23], and the two thyroid laminas have almost met medially to fuse [13]. The fetus performs reflexive actions, but no sensory fibers extend to the epithelium in most areas of the pharynx and larynx [10]. An adult pattern of motor innervation is present [10] with the cricopharyngeal muscle identified at the posteroinferior margin of the cricoid [18]. The vocalis muscle begins differentiation with muscle fibers extending toward the vocal ligament [18].
For the remainder of in utero development, few changes to the structure of the larynx occur, but laryngeal function becomes established. During week 8, the vocal folds begin their development and are observed to be a slit-like opening in the larynx [8]. During week 9, the ventral borders of the thyroid lamina begin to meet with fusion completed by week 12 [6, 13]. The true and false vocal folds are differentiated around the laryngeal ventricles between weeks 10 and 12 [13, 19]. During week 11, random “flutter-like” movement of the glottis is noted through ultrasound, becoming more consistent by week 17 [24]. During week 15, the epiglottis has descended to the level of the thyroid [25]. Between weeks 17 and 20, the epiglottis is noted to contain fibroelastic cartilage [6]. During week 21, the epiglottis is in near apposition to the uvula [26]. The free borders of the VF contain one to two cell layers of the nonkeratinized squamous epithelium [27]. The cuneiforms appear during week 26 [28]. The epiglottis takes on its omega shape between weeks 29 and 32 [6]. The membranous portion of the vocal folds contains stratified squamous epithelium and is differentiated from the cartilaginous portion by week 30 and distinct from the cartilaginous portion of the vocal folds by 38 weeks [22] with glottic movement decreasing leading up to birth [20].
Human Embryonic Feeding and Swallowing
Swallowing function is the first motor response to develop and can be seen as early as week 11, coinciding with taste bud development as well as the jaw-opening reflex and beginning signs of tongue activity [29, 30]. During week 15, anti-regurgitation reflex mechanisms appear [31] as well as pre-feeding skill development of inconsistent tongue thrust and non-nutritive suck/swallow [24, 29]. Fetuses have been noted to swallow approximately 18–50 ml/kg of amniotic fluid daily by week 16 [32, 33] as well as demonstrate inconsistent tongue cupping [24]. During week 18, the fetus has inconsistent tongue protrusion with sucking movements identified between weeks 18 and 20 [24, 30]. Myelination of CNs III, IV, VI, VII, IX, and XII occurs with the appearance of jaw-opening/jaw-closing movements, anterior tongue movement, and suckling between weeks 18 and 24 [29]. Through color Doppler imaging, fetuses have been identified to have an uncoordinated swallowing and regurgitation phenomena from weeks 19 to 28 [31]. During week 21, tongue thrusting becomes more consistent with a more consistent swallow developing by week 22, which is also when substantial weight gain of the fetus is appreciated [24, 29]. Fetal facial responses to bitter tastes have been noted between weeks 26 and 28 [34]. Consistent tongue cupping and tongue protrusion have been appreciated during week 28 [24]. Coordination of the swallow with a simultaneous decrease in frequency of regurgitation is noted during week 29 [31]. By weeks 35–38, the fetal nervous system is able to sufficiently carry out integrative functions, such as nipple feeding, with near-term fetuses swallowing amniotic fluid at a rate of 500–1000 ml/day [29, 33].
Human Postnatal Development
At birth, the thyroid cartilage and hyoid bone are attached to one another. Over time, as the larynx descends, the two become separated. At birth, the larynx sits around cervical vertebrae C1–C4 to promote the suck-swallow-breathe phenomena seen in young infants [26, 35, 36]. At 4–6 months of age, infants begin to transition to use of oral respiration coinciding with changes to the neuromuscular control of the larynx and pharynx [26]. At 2 years of age, the larynx is situated between C2 and C5 [26], and by age 5, the larynx sits around C5 [35, 36]. This pattern of descent continues until it reaches its final position at C7 between the ages of 15 and 20 [31, 35]. The cartilages also begin to ossify as one ages with the hyoid beginning at age 2 [36]. Ossification does not begin again until one reaches their 20s when the thyroid and cricoid cartilages begin to ossify [36]. The ossification process is complete by the age of 65, apart from the cuneiform and corniculate cartilages [36].
Changes to the layered structure of the vocal folds occur postnatally. At birth, the vocal folds are a hypercellular monolayer at the level of the lamina propria with single cells randomly distributed and of various shapes with traces of elastin [37]. They are 2.5–3.0 mm in length [38]. By about 2 months of age, signs of cellular differentiation and progression to a bilaminar structure can be seen [37]. There is a hypocellular superficial layer with a deeper layer containing plumper and less spindly shaped cells. By 11 months of age, a three-layered structure can be seen developing based on the various cell densities [37]. There continues to be a hypocellular layer beneath the epithelial cover, the medial layer that is more hypercellular, and the deepest layer that is just superficial to the vocalis and is also hypocellular [37]. A three-layered structure is clearly defined by the age of 7, with identifiable distinct regions of cell density [37]. The superficial layer continues to remain hypocellular, the medial layer is denser with an increase in cellularity and a region with increased amounts of collagen and elastin, and the deepest layer becomes less cellular [37]. At age 10 years, the vocal folds lengthen from approximately 6–8 mm to about 8.5–12 mm in females and 14.5–18 mm in males. By ages 11 and 12 years, we can see the classic pattern of the lamina propria, containing a hypocellular superficial layer, elastin prominent intermediate layer, and collagen prominent deep layer [37].
The timing of full maturation of the layered structure of the vocal folds coincides with when humans reach puberty. In males, this is perceptually noted as a lower-pitched voice. Research has shown that this is due to the increase in testosterone which thickens the laryngeal cartilages and vocal folds [38]. This is the first of many differences between male and female laryngeal anatomy. In addition to pitch differences, men are noted to have a larger thyroid lamina, an acute thyroid angle (creating the large “Adam’s apple”), and larger glottal space [38]. Women on the other hand are noted to have a larger posterior cartilaginous space which has been hypothesized to contribute to how women use their voices culturally giving the perception of a breathy voice [38].
Murine Laryngeal and Vocal Fold Development
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