# Is there geometric morphometric evidence for the selection against impaction? A comparative cross-sectional study of specific tooth-agenesis patterns

Sarah Boussali, Anaïs Cavaré

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Sarah Boussali, Anaïs Cavaré. Is there geometric morphometric evidence for the selection against impaction? A comparative cross-sectional study of specific tooth-agenesis patterns. Archives of Oral Biology, 2025, 182, pp.106462. 10.1016/j.archoralbio.2025.106462. hal-05461200

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# Is there geometric morphometric evidence for the selection against impaction? A comparative cross-sectional study of specific tooth-agenesis patterns

Sarah Boussali<sup>a</sup>, Anaïs Cavaré<sup>a,b,*</sup>

## ARTICLE INFO

**Keywords:**

Tooth agenesis

Hypodontia

Morphogenesis

Morphometrics

Tooth development

Craniofacial shape

Human evolution

## ABSTRACT

**Objectives:** Reductions in tooth number and facial prognathism may reflect shared genetic and evolutionary mechanisms but have been inconsistently reported by traditional cephalometric analyses. This study aimed to assess whether common patterns of dental agenesis are associated with craniofacial morphology using geometric morphometric methods.

**Design:** This retrospective comparative cross-sectional study included 538 patients aged 10–19 years from a French orthodontic population. Individuals with hypodontia (n = 269), restricted to third molars, maxillary lateral incisors, or second premolars, were matched by age and sex with controls without agenesis (n = 269). Craniofacial morphology was assessed on lateral cephalograms using 18 landmarks and 87 semilandmarks. Multiple linear regression and Procrustes MANOVA were applied to evaluate differences in size and shape, adjusting for sex and age.

**Results:** No significant association was found between agenesis status and craniofacial size in the overall configuration as well as in the cranial base, maxilla, and mandible. A significant sex effect was detected in the mandible, with smaller centroid size in males (p = 0.012). Multivariate regression on shape confirmed allometric effects across all configurations. Procrustes MANOVA detected a significant effect of sex on overall shape (p = 0.003), but neither age nor hypodontia reached significance.

**Conclusion:** In this large geometric morphometric study, no generalized or localized alterations in craniofacial morphology were detected, even in third molar agenesis, suggesting that the anthropological significance of hypodontia should be regarded with caution.

## 1. Introduction

Tooth agenesis, defined as the congenital absence of one or more permanent teeth, is among the most common developmental dental anomalies in anatomically modern humans. Depending on whether third molars are included, meta-analyses have reported prevalence rates ranging from 6.4% to over 22% (Carter & Worthington, 2015; Khalaf et al., 2014). The teeth most frequently missing are located at the distal ends of the dental series, and recent evidence suggests that third molar agenesis should not be considered in isolation but rather as part of a continuum within the same genetic disorder as hypodontia (Cavaré et al., 2025).

Notably, dental agenesis has been associated with mutations in

several genes, including MSX1, PAX9, and WNT10A, which play dual roles in odontogenesis and craniofacial morphogenesis (Nassif et al., 2014; Shaffer et al., 2016; Vieira et al., 2004; Zhang et al., 2023). These overlapping genetic pathways suggest that dental agenesis may not be an independent trait from craniofacial morphology. This view is consistent with classical anthropological theories, which associate the reduction in tooth size and number with a craniofacial morphological trend toward facial flatness (Lacy, 2021; Trinkaus, 2003). Since the transition to agriculture, cultural shifts toward softer diets have reduced masticatory stress, limiting jaw growth and the space available for late-forming dental germs (Corruccini, 1984; Galland et al., 2016; Katz et al., 2017). The “selection against impaction” theory suggests that natural selection may have contributed to third molar agenesis as a

<footnote><sup>a</sup> Université de Bordeaux, UFR des Sciences Odontologiques, Bordeaux, France</footnote>

<footnote><sup>b</sup> Université de Bordeaux, CNRS, MCC, PACEA, UMR 5199, Pessac, France</footnote>

<footnote>* Corresponding author at: Université de Bordeaux, UFR des Sciences Odontologiques, Bordeaux, France.</footnote>

<footnote>E-mail address: anais.cavare@u-bordeaux.fr (A. Cavaré).</footnote>
response to impaction, thereby avoiding severe consequences in the absence of adequate dental care (Brothwell et al., 1963; Carter, 2016).

To date, a substantial amount of research using traditional cephalometric analyses has investigated the association between tooth agenesis and craniofacial skeletal characteristics, reporting reduced anterior facial height, shorter mandibular bodies, smaller mandibular plane angles, and a tendency toward skeletal Class III patterns (Brézulier et al., 2024; Endo et al., 2006). Despite some conflicting reports concerning the presence or magnitude of the morphological effects (Ramiro-Verdugo et al., 2015; Yelmer & Akbulut, 2022), two recent meta-analyses provide overall support for these observations. Rodrigues et al. (2020) reported that agenesis—particularly of anterior or multiple teeth—is associated with a more retruded skeletal profile, reflected in reduced SNA and ANB angles, while Le et al. (2024) confirmed that patients with third molar agenesis exhibit a shorter maxillary length and a brachyfacial skeletal pattern. As third molars show the highest incidence of agenesis, understanding their craniofacial associations is particularly relevant from both developmental and clinical perspectives. These relationships may reflect broader growth tendencies with implications for orthodontic diagnosis and early intervention, especially since brachyfacial Class III skeletal patterns often require early orthopedic management, carry a less favorable prognosis, and demand long-term follow-up.

However, traditional morphometric approaches, based on linear and angular measurements, provide only limited information on craniofacial shape, as they are often redundant, highly correlated, and dominated by size (Halazonetis, 2004; Moyers & Bookstein, 1979; Zelditch et al., 2012). Their reliance on mostly univariate analyses can yield misleading interpretations of subtle or complex morphological variation. To address these shortcomings, recent studies have increasingly adopted geometric morphometric methods (GMM), which allow a more robust assessment of shape variation, independent of size, position, and orientation (Bookstein, 1989, 1992). Applied to hypodontia, GMM studies have provided new insights into size and shape variation, consistently showing reduced facial size (Gkantidis et al., 2021a; Oeschger et al., 2020a) and flatter profiles (Kanavakis et al., 2024; Oeschger et al., 2022), although results remain inconsistent for isolated third molar agenesis (Cocos & Halazonetis, 2017).

Most of this evidence derives from overlapping populations and emphasizes the number of missing teeth, an interpretation not fully consistent with theories of natural selection against impaction. Further research is needed to validate existing findings and to uncover new patterns of morphological variation using this approach. By investigating a large, geographically distinct sample, we aim to clarify the role of third molars as a suitable model for studying this anthropological hypothesis, and to extend this investigation to two other commonly missing teeth considered in isolation, since it remains unclear whether similar selective pressures also apply to other forms of tooth agenesis.

Therefore, the objective of this study is to assess the association between dental agenesis and craniofacial morphology using geometric morphometrics analyses of lateral cephalometric radiographs, and to contribute to our understanding of the evolutionary trajectory of dentition and facial form.

## 2. Materials and methods

This study follows the recommendations provided by the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for presenting observational studies (Vandenbroucke et al., 2014). Approval was obtained from the Research Ethics Committee of the University Hospital of Bordeaux (CER-BDX 2023–133). Written consent was obtained from the child's legally responsible carer, and data were anonymised twice.

### 2.1. Study design and participants

This retrospective comparative study was conducted on a pre-existing sample (Cavare et al., 2024; Cavaré et al., 2025) obtained from orthodontic patient records over an 18-year period (2006–2024) in a private practice in southwestern France. In geometric morphometrics, no standard method currently allows for a priori sample size estimation based on mean Procrustes distance, in part due to variability in landmark configurations and the high dimensionality of the data (Cardini et al., 2015). Moreover, although recent studies on the subject of this study often report mean differences in centroid size, they frequently omit measures of dispersion, thereby limiting the estimation of effect sizes and the sample size required to detect biologically meaningful differences (Gkantidis et al., 2021b; Oeschger et al., 2020b). To address this limitation and ensure adequate statistical power, we included all available patients who met the predefined inclusion and exclusion criteria in the affected group.

Eligible participants were selected according to the following inclusion criteria:

- Availability of a complete medical record

- Age between 10 and 19 years at the time of lateral cephalometric acquisition

- Congenital absence of at least one permanent tooth, restricted to third molars, maxillary lateral incisors, or second premolars

- High-quality lateral cephalometric radiograph in maximum intercuspation, with a visible calibration scale

- Good-quality panoramic radiograph obtained prior to treatment, allowing clear identification of missing teeth. Because delayed mineralization of second premolars and third molars has been reported (Dhamo et al., 2016), any diagnosis of agenesis involving these teeth in patients younger than 12 years required confirmation on a subsequent panoramic radiograph.

The following exclusion criteria were applied:

- Incomplete records, including missing or poor-quality imaging

- History of dental extraction

- Prior orthodontic treatment or any intervention likely to affect craniofacial morphology

- Presence of systemic diseases or syndromes known to influence craniofacial growth

- Combined agenesis involving both third molars and other permanent teeth.

This sample was matched with a control group of unaffected children of the same sex, age, and geographic origin, randomly selected among individuals meeting the same radiographic and medical record quality criteria. The hypodontia group was further divided into subgroups to explore the specific craniofacial effects associated with different agenesis patterns.

The final study sample consisted of 538 participants who met the above criteria, including 269 individuals with hypodontia and 269 matched controls. For each participant, the type and number of congenitally missing teeth (CMT) were recorded, along with sex and age at the time of lateral cephalometric radiograph acquisition. The FDI World Dental Federation notation system was used to identify individual teeth. The distribution of these characteristics within each group is summarized in Table 1. Because the study considered each agenesis pattern separately, the mean number of congenitally missing teeth varied between 1.4 and 2 across subgroups (range: 1–4 CMT).

### 2.2. Image acquisition and sampling

All lateral cephalometric radiographs were digitized at high resolution (minimum 300 dpi) in JPEG format and imported into Image J
<table><caption>**Table 1**
Baseline characteristics of study participants in the control and agenesis groups.</caption><thead><tr><th>Characteristic</th><th>Agenesis</th><th>Control</th><th>p-value<sup>b</sup></th></tr></thead><tbody><tr><td>Age (years)</td><td>N = 269<sup>a</sup><br>13.11 ± 1.54</td><td>N = 269<sup>a</sup><br>13.11 ± 1.29</td><td>0.6</td></tr><tr><td>Sex</td><td></td><td></td><td>&gt; 0.9</td></tr><tr><td>Female</td><td>161 (60 %)</td><td>161 (60 %)</td><td></td></tr><tr><td>Male</td><td>108 (40 %)</td><td>108 (40 %)</td><td></td></tr><tr><td>Patterns of CMT</td><td></td><td></td><td></td></tr><tr><td>No CMT</td><td>-</td><td>269 (100 %)</td><td></td></tr><tr><td>M3</td><td>151 (56 %)</td><td>-</td><td></td></tr><tr><td>PM2</td><td>74 (28 %)</td><td>-</td><td></td></tr><tr><td>ULI</td><td>44 (16 %)</td><td>-</td><td></td></tr></tbody></table>

CMT: congenitally missing teeth; M3: third molar agenesis; PM2: second pre-molar agenesis; ULI: upper lateral incisor agenesis

software (Schneider et al., 2012). A calibration was performed on each image using a reference ruler of known dimensions included in the radiograph. The sampling of digitised patient lateral cephalographs was

conducted by a single trained examiner (Author 1) between January and April 2025. The main data consisted of a combination of 18 anatomical landmarks and 87 curve semilandmarks, placed on craniofacial structures using the “Multipoint” tool in ImageJ (Fig. 1, Table 2). Landmark selection was guided by previous studies on morphological variation of the mandible, maxilla, or cranial base (Alamoudi et al., 2024; Chu et al., 2023; Oeschger et al., 2020b; Xiong et al., 2023). Semilandmarks were allowed to slide along their respective curves to minimize bending energy, using an iterative procedure implemented during a subsequent step of the Generalized Procrustes Analysis (Bookstein, 1989; Gunz et al., 2005; Gunz & Mitteroecker, 2013). The Cartesian coordinates were then exported as “.txt” files, labeled with an anonymized identification number, for further analysis.

## 2.3. Geometric morphometrics analyses

For each anatomical structure (cranial base, mandible, maxilla, and full craniofacial complex), a Generalized Procrustes Analysis or GPA (Rohlf, 1999; Slice, 2001) was independently conducted using the gpagen function from the geomorph package (Adams & Otárola-Castillo,

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Digitization of lateral cephalographs using 18 anatomical landmarks (labelled) and 87 curve semilandmarks. Cranial base structures are shown in yellow, maxillary structures in purple, and mandibular structures in blue. The full set represents the complete craniofacial configuration.</figcaption></figure>

<footnote><sup>a</sup> Mean ± SD; n (%)</footnote>

<footnote><sup>b</sup> Wilcoxon rank sum test; Pearson's Chi-squared test</footnote>
<table><caption>**Table 2**
Fixed cephalometric landmarks used in the geometric morphometric analysis.</caption><thead><tr><th>Name</th><th>Description</th></tr></thead><tbody><tr><td>F.cæcum</td><td>Foramen caecum - the midline aperture between the crista galli and frontal bone</td></tr><tr><td>ACP</td><td>Anterior clinoid process - the posterior projection of the sphenoid bone at the junction of the medial end of either lesser wing of sphenoid bone with the body of sphenoid bone</td></tr><tr><td>PCP</td><td>Posterior clinoid process - the bony prominence located at the superolateral aspect of the dorsum sellae</td></tr><tr><td>Ba</td><td>Basion - The lowest point on the anterior margin of the foramen magnum in the median plane</td></tr><tr><td>DCL/S</td><td>Point of intersection between the De Coster line and the greater wing of the sphenoid</td></tr><tr><td>Pr</td><td>Prosthion - The point of the upper alveolar process that projects most anteriorly</td></tr><tr><td>A</td><td>A-Point - The deepest point on the contour of the maxillary alveolar process</td></tr><tr><td>ANS</td><td>Anterior nasal spine</td></tr><tr><td>PNS</td><td>Posterior nasal spine</td></tr><tr><td>Mx Pl</td><td>Mx Posterior Infradentale - The intersection of the alveolar bone of the maxilla with the palatal surface of the maxillary incisor.</td></tr><tr><td>Ar</td><td>Articulare - The point of intersection of the external dorsal contour of the mandibular condyle and the temporal bone</td></tr><tr><td>Go</td><td>Gonion - The most posterior and inferior point on the bony chin</td></tr><tr><td>Me</td><td>Menton - The most inferior point of the mandibular symphysis</td></tr><tr><td>Pg</td><td>Pogonion - The most prominent point of the mandibular symphysis</td></tr><tr><td>B</td><td>B-Point Deepest point on the curved profile of the mandible between the chin and alveolar crest</td></tr><tr><td>Mn Al</td><td>Mn Anterior Infradentale - Highest anterior point of the mandibular alveolar bone</td></tr><tr><td>Mn Pl</td><td>Mn Posterior Infradentale - Highest posterior point of the mandibular alveolar bone</td></tr><tr><td>Pg</td><td>Pogonion' - The most prominent point of posterior symphysis</td></tr></tbody></table>

2013; Baken et al., 2021) in R version 4.5.1. This procedure standardizes landmark configurations by removing differences in position, orientation, and scale, performing a superimposition of all specimens using a least-squares criterion that minimizes distances between homologous landmarks. The resulting Procrustes shape coordinates served as the input for shape analysis. Potential outliers were identified by inspecting the distribution of Procrustes distances from the mean shape. Specimens with values exceeding the interquartile range were carefully re-examined to identify digitization errors.

Size was investigated using the natural logarithm of centroid size (lnCS), a reliable proxy for overall structural size that is independent of shape. Centroid size corresponds to the square root of the sum of squared Euclidean distances between each landmark and the configuration's centroid, and was computed during coordinate normalization performed through GPA (Mitteroecker & Gunz, 2009).

## 2.4. Statistical analysis

To examine whether size differed according to hypodontia, sex, or age, a multiple linear regression was performed with lnCS as the dependent variable. If a significant effect of agenesis was detected, pairwise comparisons between agenesis sub-groups were planned to assess specific differences in size, with p-values adjusted for multiple testing. Boxplots were used to support the interpretation of statistical comparisons.

To assess the presence of allometry, a multivariate regression of Procrustes shape coordinates on lnCS was first conducted using the procD.lm() function from the geomorph package. Statistical significance was evaluated based on 1 000 permutation replicates to account for the non-independence of shape variables. As the primary aim of this study was to evaluate the impact of dental agenesis and potential confounding variables such as age and sex, rather than to analyze shape variation related to allometric size, the residuals from the regression were retained for further analyses.

These residuals, representing shape variation independent of size, were subjected to an exploratory principal component analysis (PCA) to reduce dimensionality and identify the main axes of shape variation. Morphological changes along the principal components were visualized using thin-plate spline (TPS) deformation grids by warping the mean shape along each PC axis (Bookstein, 1989, 1992). This approach facilitated the interpretation of shape differences among groups and helped identify biologically meaningful patterns of morphological variation. To further explore group discrimination between agenesis patterns and controls, a between-group principal component analysis (bgPCA) was performed using the Morpho package (Schlager, 2017).

Then, for inferential testing, we used the regression residuals as the dependent variables in a multivariate analysis of variance (MANOVA), with 1 000 permutations for significance testing, performed separately for the cranial base, maxilla, mandible, and the overall craniofacial structure. These models aimed to assess whether craniofacial shape differed significantly according to dental agenesis status, while controlling for sex and age as potential confounding factors. The relationship between age and size was assessed using Spearman's rank correlation coefficient, given that lnCS was not normally distributed (Shapiro-Wilk test, p = 0.03), which revealed no significant association (p = 0.016, p = 0.714). Given this result, age was retained as a separate variable in these analyses, as its influence on shape was considered independently of size.

Finally, we performed pairwise comparisons using a Randomized Residual Permutation Procedure (RRPP) with 1000 permutations to examine differences in mean shape among agenesis patterns (Collyer & Adams, 2018). P-values were adjusted for multiple comparisons using the False Discovery Rate (FDR) correction method.
## 2.5. Measurement error

Intra-observer reliability in landmark placement was evaluated by the principal investigator (Author 1), who repeated the digitization of four randomly selected specimens ten times each using ImageJ. The resulting coordinate datasets were subjected to GPA, followed by a PCA on the Procrustes shape coordinates to visually assess the dispersion of repeated measurements in shape space. To quantify digitization error, the measurement.error() function from the geomorph package was also used (Collyer & Adams, 2024). This procedure performs a Procrustes ANOVA (Goodall, 1991) to partition total shape variance into components attributable to inter-individual variation, systematic error (variation among repeated measurements of the same specimen), and random digitizing error. It also computes an intra-class correlation coefficient (ICC) as a measure of repeatability. The interpretation of the ICC was based on the scale proposed by Landis and Koch (Landis & Koch, 1977).

# 3. Results

## 3.1. Measurement error

The intra-class correlation coefficient (ICC) assessing measurement agreement was 0.88, indicating excellent intra-observer reliability in landmark placement. Most of the variation was attributable to individual differences, with no significant contribution from systematic measurement error (Table 3). The PCA revealed clustering of repeated digitizations for each individual in shape space, indicating that intra-observer variation was negligible compared to biological variability (Fig. 2). The first two principal components together accounted for nearly 70 % of the total shape variance.

## 3.2. Size analysis

Across all configurations, no significant association was observed with agenesis status or age (Table 4). However, a significant effect of sex was detected in the mandibular configuration, with smaller size observed in males (p = 0.012). To support the interpretation of these findings, boxplots illustrating the distribution of lnCS by agenesis patterns are provided for the overall craniofacial configuration in Fig. 3, and separately for each anatomical region in Supplementary Fig. S1.

## 3.3. Allometry

The multivariate regression analyses revealed a significant allometric effect across all anatomical configurations (Table 5), with lnCS explaining between 1.5 % and 3.6 % of the shape variance.

## 3.4. Shape analysis

Shape variation along the principal components reflects subtle morphological changes, which are not always easily perceptible in the deformation grids without magnification (Fig. 4; Supplementary Figures S2–S5). To facilitate visual interpretation, magnified grids (×3) are provided in Supplementary Figure S6. In the overall configuration,

<table><caption>**Table 3**

Results of ProcANOVA evaluating systematic and random measurement error in landmark digitization.</caption><thead><tr><th></th><th>Df<sup>a</sup></th><th>Sum of Squares</th><th>R<sup>2</sup></th><th>p-value</th></tr></thead><tbody><tr><td>Subjects</td><td>3</td><td>0.216</td><td>86.66 %</td><td><strong>0.001</strong></td></tr><tr><td>Systematic Measurement Error</td><td>9</td><td>0.009</td><td>3.69 %</td><td>0.142</td></tr><tr><td>Random Measurement Error</td><td>27</td><td>0.024</td><td>9.65 %</td><td></td></tr><tr><td><strong>Total</strong></td><td>39</td><td>0.249</td><td></td><td></td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Principal Component Analysis (PCA) of Procrustes coordinates to assess measurement error. Each colored dot represents the Procrustes coordinates of a given specimen across repeated digitizations.</figcaption></figure>

<table><caption>**Table 4**

Multiple linear regression of centroid size on agenesis status, sex, and age.</caption><thead><tr><th>Configurations</th><th>Parameters</th><th>β coefficient</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="4">Overall craniofacial</td><td>Intercept</td><td>mean</td><td>± SE<sup>a</sup></td></tr><tr><td>Agenesis</td><td>6.027</td><td>0.020</td></tr><tr><td>Sex (Male)</td><td>-0.001</td><td>0.004</td></tr><tr><td>Age</td><td>-0.004</td><td>0.004</td></tr><tr><td rowspan="4">Cranial base</td><td>Intercept</td><td>mean</td><td>± SE<sup>a</sup></td></tr><tr><td>Agenesis</td><td>5.140</td><td>0.021</td></tr><tr><td>Sex (Male)</td><td>-0.005</td><td>0.005</td></tr><tr><td>Age</td><td>-0.008</td><td>0.005</td></tr><tr><td rowspan="4">Maxilla</td><td>Intercept</td><td>mean</td><td>± SE<sup>a</sup></td></tr><tr><td>Agenesis</td><td>4.300</td><td>0.032</td></tr><tr><td>Sex (Male)</td><td>-0.003</td><td>0.007</td></tr><tr><td>Age</td><td>-0.001</td><td>0.007</td></tr><tr><td rowspan="4">Mandible</td><td>Intercept</td><td>mean</td><td>± SE<sup>a</sup></td></tr><tr><td>Agenesis</td><td>5.200</td><td>0.027</td></tr><tr><td>Sex (Male)</td><td>-0.006</td><td>0.006</td></tr><tr><td>Age</td><td>-0.014</td><td>0.006</td></tr></tbody></table>

PC1 described a transition from a downward positioned mandible with an open gonial angle, reduced facial convexity, and a straighter cranial base to an anteriorly displaced maxilla with reduced vertical dimension and a more acute gonial angle (Fig. 4). PC2 illustrated a shift from a forward position of point A with reduced palatal height and a robust symphysis to a taller, more gracile mandible with a retruded point B and a downward-inclined mandibular body. The distribution of individuals in shape space along the first two principal components, which together accounted for 34.7 % of the total variance, showed extensive overlap between groups. No clear separation was observed between controls and the different agenesis patterns, although individuals with premolar agenesis tended to shift slightly toward lower PC2 values, without forming distinct clusters. The next two components (PC3 and PC4) accounted for an additional 17 % of the variance but similarly did not reveal any clear group separation (Supplementary Fig. S2). The bgPCA provided a supervised ordination emphasizing group differences while minimizing the influence of other factors such as sex-related variation. While it suggested a partial separation of subjects with second premolar or upper lateral incisor agenesis from controls and third molar agenesis, substantial overlap remained across groups, indicating that inter-group differences were subtle compared to intra-group variability.

When analysed separately, each configuration showed extensive group overlap across the first four PCs as well as in the bgPCA, with no clear clustering emerging from either conventional or supervised

<footnote>a Df: degrees of freedom</footnote>

<footnote>a SE: standard error</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Boxplot of size (lnCS) by sex for the overall craniofacial configuration, according to dental agenesis patterns (M3: third molar agenesis; PM2: second premolar agenesis; ULI: upper lateral incisor agenesis).</figcaption></figure>

<table><caption>**Table 5**
Allometric effects assessed by multivariate regression of shape on centroid size.</caption><thead><tr><th>Configurations</th><th>Df<sup>a</sup></th><th>SS<sup>b</sup></th><th>R<sup>2</sup></th><th>F</th><th>Z</th><th>p-value</th></tr></thead><tbody><tr><td>Overall craniofacial</td><td>1</td><td>0.098</td><td>3.64 %</td><td>20.225</td><td>6.420</td><td><strong>0.001</strong></td></tr><tr><td>Cranial base</td><td>1</td><td>0.061</td><td>1.53 %</td><td>8.325</td><td>5.377</td><td><strong>0.001</strong></td></tr><tr><td>Maxilla</td><td>1</td><td>0.111</td><td>1.58 %</td><td>8.580</td><td>3.578</td><td><strong>0.001</strong></td></tr><tr><td>Mandible</td><td>1</td><td>0.059</td><td>2.70 %</td><td>14.847</td><td>5.426</td><td><strong>0.001</strong></td></tr></tbody></table>

ordination (Supplementary Fig. S3-S5). For the cranial base, the overall pattern of shape variation was broadly consistent with the isolated analysis, with PC1 reflecting a transition from a straighter and elongated base to a relatively shortened and flexed one (Supplementary Fig. S3). By contrast, PC2 in the isolated analysis revealed additional local

changes not evident in the global configuration, namely a flexion of the basi-occipital region and a deepening of the sella turcica in the sphenoidal body. For the maxilla (Supplementary Fig. S4), the isolated analysis confirmed the global trend but further highlighted a flatter posterior palate with greater anterior alveolar bone thickness (maximum of PC1) and an increase in anterior alveolar height (maximum of PC2). For the mandible, the isolated analysis partly contrasted with the overall configuration, with PC1 showing greater variation in symphyseal shape and PC2 revealing vertical changes, including the appearance of a pregonial notch and an open gonial angle at higher values (Supplementary Fig. S5). On average, premolar agenesis remained close to controls, whereas third molar and upper lateral incisor agenesis exhibited comparable shifts in maxillary conformation along PC1 but diverged in the mandible, with M3 occupying lower and ULI higher PC1 values. Additional descriptions of shape variation along PC3

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Principal Component Analysis (PCA, left) and between-group Principal Component Analysis (bgPCA, right) of Procrustes shape coordinates in the overall configuration. Shape hulls and colours distinguished the individuals by their agenesis patterns. Thin-plate spline deformation grids illustrate shape variation along the main axes, without magnification to preserve biological interpretation.</figcaption></figure>

<footnote><sup>a</sup> Df: degrees of freedom; <sup>b</sup> SS: sums of squares</footnote>
and PC4 for the overall configuration, the cranial base, the maxilla, and the mandible are provided in the **Supplementary Material**.

The results of Procrustes MANOVA and pairwise comparisons for each configuration are presented in Table 6. In the overall craniofacial configuration, General Linear Models showed a significant effect of sex on shape variation (F= 3.2345, p = 0.003), whereas neither age nor agenesis status reached significance. When interactions were included in

<table><caption>**Table 6**

Results of Procrustes MANOVA and RRPP pairwise comparisons for the effects of age, sex, and agenesis patterns on shape across configurations.</caption><thead><tr><th colspan="10">Overall craniofacial</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr><tr><th>MANOVA</th><th>Df<sup>a</sup></th><th></th><th>F</th><th>p-value</th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Sex</td><td>1-537</td><td>-</td><td>-</td><td>3.2345</td><td><strong>0.003</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Age</td><td>1-537</td><td>-</td><td>-</td><td>1.3683</td><td>0.159</td><td></td><td></td><td></td><td></td></tr><tr><td>Agenesis<sup>d</sup></td><td>1-537</td><td>-</td><td>-</td><td>1.3427</td><td>0.176</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Pairwise comparison RRPP</strong></td><td></td><td>d<sup>b</sup></td><td>UCL (95 %)<sup>c</sup></td><td>Z</td><td>p-value</td><td>P-adj (FDR)</td><td></td><td></td><td></td></tr><tr><td>Control vs M3</td><td>-</td><td>0.0066</td><td>0.0095</td><td>-0.0991</td><td>0.533</td><td>0.640</td><td></td><td></td><td></td></tr><tr><td>Control vs PM2</td><td>-</td><td>0.0134</td><td>0.0122</td><td>2.1049</td><td><strong>0.017</strong></td><td>0.102</td><td></td><td></td><td></td></tr><tr><td>Control vs ULI</td><td>-</td><td>0.0108</td><td>0.0150</td><td>-0.0142</td><td>0.517</td><td>0.640</td><td></td><td></td><td></td></tr><tr><td>M3 vs PM2</td><td>-</td><td>0.0131</td><td>0.0131</td><td>1.6327</td><td>0.051</td><td>0.153</td><td></td><td></td><td></td></tr><tr><td>M3 vs ULI</td><td>-</td><td>0.0101</td><td>0.0161</td><td>-0.6864</td><td>0.760</td><td>0.760</td><td></td><td></td><td></td></tr><tr><td>PM2 vs ULI</td><td>-</td><td>0.0143</td><td>0.0178</td><td>0.5874</td><td>0.280</td><td>0.560</td><td></td><td></td><td></td></tr><tr><td><strong>Cranial base</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MANOVA</td><td>Df<sup>a</sup></td><td></td><td>F</td><td>p-value</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sex</td><td>1-537</td><td>-</td><td>-</td><td>2.2650</td><td>0.159</td><td></td><td></td><td></td><td></td></tr><tr><td>Age</td><td>1-537</td><td>-</td><td>-</td><td>1.7019</td><td>0.115</td><td></td><td></td><td></td><td></td></tr><tr><td>Agenesis<sup>d</sup></td><td>1-537</td><td>-</td><td>-</td><td>1.1474</td><td>0.189</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Pairwise comparison RRPP</strong></td><td></td><td>d<sup>b</sup></td><td>UCL (95 %)<sup>c</sup></td><td>Z</td><td>p-value</td><td>P-adj (FDR)</td><td></td><td></td><td></td></tr><tr><td>Control vs M3</td><td>-</td><td>0.0120</td><td>0.0120</td><td>1.6827</td><td><strong>0.047</strong></td><td>0.282</td><td></td><td></td><td></td></tr><tr><td>Control vs PM2</td><td>-</td><td>0.0096</td><td>0.0152</td><td>-0.4978</td><td>0.687</td><td>0.687</td><td></td><td></td><td></td></tr><tr><td>Control vs ULI</td><td>-</td><td>0.0143</td><td>0.0190</td><td>0.3300</td><td>0.377</td><td>0.472</td><td></td><td></td><td></td></tr><tr><td>M3 vs PM2</td><td>-</td><td>0.0125</td><td>0.0165</td><td>0.3300</td><td>0.393</td><td>0.472</td><td></td><td></td><td></td></tr><tr><td>M3 vs ULI</td><td>-</td><td>0.0163</td><td>0.0202</td><td>0.6321</td><td>0.269</td><td>0.472</td><td></td><td></td><td></td></tr><tr><td>PM2 vs ULI</td><td>-</td><td>0.0196</td><td>0.0225</td><td>1.0736</td><td>0.152</td><td>0.456</td><td></td><td></td><td></td></tr><tr><td><strong>Maxilla</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MANOVA</td><td>Df<sup>a</sup></td><td></td><td>F</td><td>p-value</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sex</td><td>1-537</td><td>-</td><td>-</td><td>2.2650</td><td>0.064</td><td></td><td></td><td></td><td></td></tr><tr><td>Age</td><td>1-537</td><td>-</td><td>-</td><td>1.7019</td><td>0.158</td><td></td><td></td><td></td><td></td></tr><tr><td>Agenesis<sup>d</sup></td><td>1-537</td><td>-</td><td>-</td><td>1.1474</td><td>0.288</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Pairwise comparison RRPP</strong></td><td></td><td>d<sup>b</sup></td><td>UCL (95 %)<sup>c</sup></td><td>Z</td><td>p-value</td><td>P-adj (FDR)</td><td></td><td></td><td></td></tr><tr><td>Control vs M3</td><td>-</td><td>0.0145</td><td>0.0178</td><td>1.0368</td><td>0.165</td><td>0.748</td><td></td><td></td><td></td></tr><tr><td>Control vs PM2</td><td>-</td><td>0.0109</td><td>0.0230</td><td>-0.5864</td><td>0.722</td><td>0.866</td><td></td><td></td><td></td></tr><tr><td>Control vs ULI</td><td>-</td><td>0.0153</td><td>0.0290</td><td>-0.2425</td><td>0.584</td><td>0.866</td><td></td><td></td><td></td></tr><tr><td>M3 vs PM2</td><td>-</td><td>0.0179</td><td>0.0255</td><td>0.6584</td><td>0.251</td><td>0.748</td><td></td><td></td><td></td></tr><tr><td>M3 vs ULI</td><td>-</td><td>0.0097</td><td>0.0291</td><td>-1.8981</td><td>0.972</td><td>0.972</td><td></td><td></td><td></td></tr><tr><td>PM2 vs ULI</td><td>-</td><td>0.0215</td><td>0.0325</td><td>0.3484</td><td>0.374</td><td>0.748</td><td></td><td></td><td></td></tr><tr><td><strong>Mandible</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MANOVA</td><td>Df<sup>a</sup></td><td></td><td>F</td><td>p-value</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sex</td><td>1-537</td><td>-</td><td>-</td><td>1.9175</td><td>0.067</td><td></td><td></td><td></td><td></td></tr><tr><td>Age</td><td>1-537</td><td>-</td><td>-</td><td>0.4829</td><td>0.854</td><td></td><td></td><td></td><td></td></tr><tr><td>Agenesis<sup>d</sup></td><td>1-537</td><td>-</td><td>-</td><td>0.7064</td><td>0.616</td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Pairwise comparison RRPP</strong></td><td></td><td>d<sup>b</sup></td><td>UCL (95 %)<sup>c</sup></td><td>Z</td><td>p-value</td><td>P-adj (FDR)</td><td></td><td></td><td></td></tr><tr><td>Control vs M3</td><td>-</td><td>0.0055</td><td>0.0094</td><td>-0.2801</td><td>0.610</td><td>0.970</td><td></td><td></td><td></td></tr><tr><td>Control vs PM2</td><td>-</td><td>0.0062</td><td>0.0121</td><td>-0.8057</td><td>0.796</td><td>0.970</td><td></td><td></td><td></td></tr><tr><td>Control vs ULI</td><td>-</td><td>0.0073</td><td>0.0149</td><td>-1.0028</td><td>0.835</td><td>0.970</td><td></td><td></td><td></td></tr><tr><td>M3 vs PM2</td><td>-</td><td>0.0051</td><td>0.0130</td><td>-1.8430</td><td>0.970</td><td>0.970</td><td></td><td></td><td></td></tr><tr><td>M3 vs ULI</td><td>-</td><td>0.0084</td><td>0.0160</td><td>-0.6374</td><td>0.731</td><td>0.970</td><td></td><td></td><td></td></tr><tr><td>PM2 vs ULI</td><td>-</td><td>0.0102</td><td>0.0177</td><td>-0.3077</td><td>0.632</td><td>0.970</td><td></td><td></td><td></td></tr></tbody></table>

the Procrustes MANOVA, the sex × hypodontia effect did not reach significance, supporting the validity of analysing the entire sample together (F= 0.9140; p = 0.485). In the isolated analyses of the cranial base, maxilla, and mandible, no significant effects of sex, age, or agenesis were detected. Pairwise comparisons of agenesis patterns using randomisation-based resampling with 1000 permutations indicated tendencies for differences between controls and premolar agenesis in the overall configuration, and between third molar agenesis and controls in the cranial base, but none remained significant after FDR correction. Although non-significant, the smallest inter-group distances were observed for the mandible, whereas the largest distances consistently involved upper lateral incisor and premolar agenesis across all configurations.

## 4. Discussion

Although not the primary focus of this study, an interesting finding was the presence of sexual dimorphism in craniofacial morphology. The linear regression model detected a significant sex effect on mandibular size, with males showing smaller centroid size values compared to females (p = 0.012). This contrasts with previous reports of larger mandibular dimensions in males, whether based on linear metrics (Cappella et al., 2020) or centroid size analyses (Chalazoniti et al., 2024; Toneva et al., 2023). In our study, sex differences were also evident in the shape of the overall craniofacial configuration (F=3.2345; p = 0.003), in accordance with previous reports (Cocos & Halazonetis, 2017; Kanavakis et al., 2024; Oeschger et al., 2022). Rosas and Bastir (2002) had similarly emphasized dimorphism of the viscerocranium, particularly pronounced in the mandible. In our sample, the mandibular configuration considered in isolation did not reach statistical significance for sex differences. This is not incompatible with previous forensic and anthropological observations, since recent GMM studies in 3D have shown that mandibular shape differences are subtle, and sexual dimorphism may also be expressed through differential growth rotations rather than pure mandibular form (Björk & Skieller, 1972). Alarcón et al. (2016) further highlighted that sex-specific mandibular traits behave differently across vertical facial patterns, reflecting the influence of craniofacial growth orientation, an aspect partly reflected in the overall configuration analysis of the present study.

A significant allometric effect on craniofacial shape was also detected in multivariate regression across all configurations, although the effect remained limited. In our study, no correlation between age and size was observed, as confirmed by Spearman's coefficient, and age never reached significance as a covariate in the models. One possible explanation is that we used chronological age, which does not necessarily reflect skeletal maturation, or that the observed effect primarily reflects static allometry rather than age-related growth. Static allometry in Homo sapiens has been extensively described by Rosas and Bastir (2002), who highlighted proportional shifts between the neurocranium and the face, along with rotational changes, with positive allometry producing a forward rotation of the lower face.

The main objective of this study was to investigate the association between specific agenesis patterns and craniofacial morphology. No significant size differences were detected according to the presence of hypodontia or the type of CMT in our sample. This finding suggests that agenesis may not exert a measurable effect on global craniofacial size, nor a local effect on the dimensions of the jaw affected by agenesis (for example the maxilla in cases of missing upper lateral incisor). In the aforementioned meta-analysis, Le et al. (2024) showed that maxillary length (ANS-PNS distance) in patients with third molar agenesis was smaller than in controls, and that mandibular corpus length (Go-Pog distance) was also reduced, although to a lesser extent. In contrast, Rodrigues et al. (2020) did not include a synthesis of data regarding the size of the mandibular or maxillary corpus in cases of hypodontia when third molars were not considered. Individual studies have reported significant reduction in maxillary length irrespective of the hypodontia

<footnote><sup>a</sup> Df: degrees of freedom; <sup>b</sup> d: Procrustes distance between group mean shapes; <sup>c</sup> 95 % upper confidence limit of d; <sup>d</sup> Agenesis refers to presence vs absence in MANOVA; in RRPP, subgroup comparisons were tested (M3 = third molar, PM2 = second premolar, ULI = upper lateral incisor)</footnote>
pattern (Bajraktarova-Miševska et al., 2016; Laganà et al., 2011) but several did not find any relationship between hypodontia and mandib-ular size (Ben-Bassat & Brin, 2003; Endo et al., 2006; Gungor & Turk-kahraman, 2013). Little is known about the other dimensions of the craniofacial complex in hypodontia. In the present study, centroid size was used as a proxy to summarize overall size, providing an integrated assessment across all dimensions, but limiting direct comparison with much of the existing literature. In neighboring samples, Oeschger et al. (2020) and Gkantidis et al. (2021) found reduced facial and maxillary centroid sizes in individuals with tooth agenesis, with no effects on the cranial base. The mandible, however, showed smaller centroid size only when third molar agenesis was analyzed in isolation in the latter study.

This inconsistency with our findings is also evident in the shape analysis. Visual inspection of PCA and bgPCA, together with MANOVA on Procrustes residuals, failed to detect any modification of craniofacial shape associated with hypodontia. In contrast, Oeschger et al. (2022) and Cocos and Halazonetis (2017) identified significant morphological differences between hypodontia groups (without third molars) and matched controls. When considering third molar agenesis in isolation, results have been conflicting, with some studies reporting significant craniofacial shape differences (Kanavakis et al., 2024), while others found none (Cocos & Halazonetis, 2017). Several factors may account for the differences with previous studies. The landmark configuration was largely comparable, except for the maxilla where orbital contours were excluded. The absence of significant differences in our study may partly reflect limited statistical power, since detecting subtle shape variation requires large samples; however, this explanation is unlikely, as our sample size was equivalent to those of the cited studies. Differ-ences in statistical treatment may also contribute, given that previous publications performed multivariate regressions on 80–85 % of the variance, whereas our analysis considered the total shape variance. Discrepancies may also arise from sample characteristics. Age consti-tutes an important source of variation. Our sample was restricted to participants up to 19 years old, whereas previous studies included in-dividuals in their forties or fifties, where age was often retained as a significant predictor. In older participants, dental history factors such as tooth loss or ankylosis of deciduous teeth, and restorative interventions become more prevalent and may act as confounders by altering craniofacial conformation. These effects are only partially explained by chronological age, which primarily reflects growth trajectories rather than dental status. As a result, the inclusion of older age groups may have increased the weight of hypodontia as a predictor in previous an-alyses. To our knowledge, no study has specifically addressed natural aging effects in hypodontia patients without prosthetic treatment. In addition, the longevity of infraoccluded deciduous teeth is poor in follow-up studies, particularly in cases of oligodontia (Hvaring & Bir-keland, 2020), and these teeth may compromise occlusion and vertical alveolar growth beyond skeletal maturation (Temming et al., 2024). Geographical origin may also account for some discrepancies, as our cohort was drawn from a different European population than those examined earlier. Even within Europe, genetic structuring has been shown to correlate with geographical distance, despite generally low levels of differentiation (Novembre et al., 2008).

The number of missing teeth may be critical in determining whether agenesis affects craniofacial morphology. Our study design included only patients with mild to moderate hypodontia (<4 CMT), as we adopted an anthropological perspective focused on the three patterns most prevalent in modern humans. Individuals with only a few missing teeth did not exhibit major craniofacial differences compared with un-affected controls. The literature remains controversial, but our results are more consistent with studies reporting an increasing impact of tooth number on facial form (Oeschger et al., 2020, 2022), whereas others found no effect of agenesis severity or location (Cocos & Halazonetis, 2017; Endo et al., 2006; Gungor & Turkkahraman, 2013). One possible explanation is that teeth contribute to the development of supporting structures, with craniofacial modifications from reduced occlusal

support and limited alveolar growth becoming apparent only beyond a certain threshold of agenesis. This raises questions about the extent to which dental anomalies are developmentally integrated with craniofacial form. From a genetic perspective, mild forms of agenesis may reflect minor disturbances in odontogenesis without extending to craniofacial structures, whereas more extensive patterns may arise from mutations affecting both dental and skeletal development.

By contrast, if natural selection were involved, one would expect any adaptive signal to be associated with mild forms of hypodontia that preserve function, rather than oligodontia, whose extensive tooth loss is unlikely to provide any adaptive fitness advantage. Nonetheless, specific patterns, most notably those affecting the terminal teeth of the dental series, may better support anthropological hypotheses. In particular, third molar agenesis has often been interpreted as an evolutionary adaptation of the dentition rather than a purely developmental or genetically determined anomaly, unlike other missing teeth (Galluccio & Pilotto, 2008). Nonetheless, RRPP analyses showed no significant craniofacial shape differences when comparing third molar agenesis with other agenesis patterns. At the jaw level, no differences were observed between patterns or relative to controls, suggesting that third molar absence is not directly linked to the gracilization of the mandible, an ongoing process associated with reduced masticatory load in modern populations consuming softer diets (Rando et al., 2014; von Cramon-Taubadel, 2011). In the maxilla, the reduced bone apposition in the tuberosity region described in cases of molar agenesis was not evident in our sample (Endo et al., 2006). By contrast, an unexpected result was observed at the cranial base between individuals with third molar agenesis and controls before post hoc correction, given that this region develops early and has remained relatively stable among homi-nins (Bastir et al., 2006; Bastir & Rosas, 2009). Although it did not retain statistical significance, this trend could warrant further targeted inves-tigation in larger cohorts, as comparable findings have been docu-mented (Kanavakis et al., 2024). Evidence of an association between third molar agenesis and sella turcica bridging further indicates that this region may be influenced by variation in interclinoid ligament calcifi-cation (Scribante et al., 2017).

The lack of detectable craniofacial effects highlights the need to integrate morphometric approaches with genetic data. In this regard, genome-wide association studies have so far not supported the hy-pothesis of recent positive selection for third molar agenesis, favoring neutral explanations such as genetic drift over the selection-against-impaction hypothesis (Vukelic et al., 2017).

The present study has some limitations inherent to its retrospective design, particularly the limited control over prior dental extractions, although this bias was minimized by focusing on younger participants. Another limitation concerns the assumption that our sample was rela-tively genetically homogeneous, an inference based only on geograph-ical origin. In an orthodontic context involving non-syndromic patients, it is not feasible to perform genetic profiling, which limits the inter-pretation of biological variability. As aforementioned, genetic struc-turing has been reported within European populations (Novembre et al., 2008), and a recent genome-wide study in France identified a southwest genetic cluster corresponding to the same geographical area as our study (Saint Pierre et al., 2020), suggesting that some degree of regional specificity may exist and should be taken into account when interpreting our results. The use of lateral cephalometric radiographs restricted the analysis to two dimensions, reducing the ability to detect transverse or volumetric differences that would be better assessed with three-dimensional imaging techniques. Landmark placement is a po-tential source of measurement error (Robinson et al., 2002), but our intra-observer analysis indicated that this error was negligible.

## 5. Conclusion

Our findings highlight the complexity of the relationship between hypodontia and craniofacial morphology. Rather than a uniform trait
with predictable skeletal consequences, agenesis emerges as a multi-
factorial condition shaped by genetic, developmental, and functional
factors, with effects modulated by the number of missing teeth. In mild
forms, craniofacial morphology may remain largely unaffected, under-
scoring the need for cautious clinical interpretation and individualized
treatment planning. Future research integrating larger and more diverse
cohorts, longitudinal designs, three-dimensional imaging and genetic
insights will be crucial to clarify the subtle interplay between dental
agenesis and craniofacial development.

CRediT authorship contribution statement

Sarah Boussali: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. Anaïs Cavaré: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Formal analysis, Data curation, Conceptualization.

Funding

This research received no funding.

Declaration of Competing Interest

The authors declare the absence of any conflict of interest.

Acknowledgments

The research was supported by the EVODIBIO team (PACEA, UMR 5199, University of Bordeaux). The study received financial support from the French government in the framework of the University of Bordeaux's IdEx "Investments for the Future" program/GPR Human Past.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.archoralbio.2025.106462.

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