Forensic Science International 306 (2020) 110072

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Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Estimating sex from the seven cervical vertebrae: An analysis of two

European skeletal populations
Andrew S. Rozendaala , Shelby Scottb , Tanya R. Peckmanna,* , Susan Meekc
a
Department of Anthropology, Saint Mary’s University, Halifax, Nova Scotia, B3H 3C3, Canada
b
Department of Anthropology, University of Toronto Mississauga, Mississauga, Ontario, L5L 1C6, Canada
c
Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, B3H 3C3, Canada

A R T I C L E I N F O A B S T R A C T

Article history: Methods for estimating sex from a variety of skeletal elements is vital in forensic anthropology as many of the
Received 30 June 2019 traditionally studied bones are often fragmented upon recovery. The current study examined the seven
Received in revised form 14 November 2019 cervical vertebrae to establish an accurate sex estimation method for Europeans. Discriminant functions
Accepted 18 November 2019
were developed from the seven cervical vertebrae using the maximum body height (CHT), vertebral foramen
Available online 20 November 2019
anterior-posterior diameter (CAP), and vertebral foramen transverse diameter (CTR). To date, no study has
used these variables to estimate sex from human remains. Two hundred and ninety-five adult individuals
Keywords:
(157 males,138 females) from the University of Athens and the Luis Lopes skeletal collections were studied.
Forensic anthropology population data
Adults
No population differences were exhibited between the contemporary Greek and historic Portuguese
Sex estimation skeletons, indicating that discriminant functions for sex estimation from the cervical vertebrae may not be
Cervical vertebrae population- or temporally-specific. The results of this study indicate that only CHT and CTR exhibited
Discriminant functions statistically significant sexual dimorphism. Seven multivariate discriminant functions were developed with
European accuracy rates between 80.3% and 84.5%. Cross-validation studies showed that five of the seven functions
exhibited strong statistical algorithms. This study will assist with estimating the sex of unknown Europeans
from the cervical vertebrae especially in cases where more accurate predictors of sex, such as the skull or the
pelvis, are unavailable.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction degree of osteological preservation. Sexual dimorphism of the skull

and the pelvic girdle have been extensively studied and are more
In forensic anthropology, the estimation of sex is of primary commonly used for sex estimation due to their high morphological
significance for creating a biological profile (i.e. the estimation of and metric accuracy rates across many populations; some methods
sex, age at death, stature, and ancestry) due to its dependence for reaching greater than 90% accuracy rates [5,6]. However, these
estimating skeletal age and stature [1,2]. Forensic anthropologists bones are often recovered in fragmented states as a result of
study sexually dimorphic differences by examining architectural taphonomic changes and other post-mortem damage [1,2,7,8].
and size variations through morphological and metric analyses. Therefore, developing methods of sex estimation from preserva-
Morphological methods rely on observational comparisons, tionally favoured and/or fragmentary bones is essential.
whereas metric methods apply quantitative measurements and Unlike the fragile skull or the large exposed surface areas of long
statistical algorithms to estimate whether the bone falls within the bones and the pelvic girdle, intact vertebrae are more likely to be
average male or female dimensions [3]. Metric analyses are recovered from a deposition site [9–11,14]. Research has shown
considered advantageous due to their simplicity and consistency in that the vertebral column, along with the proximal femora, is more
standardizing skeletal landmarks for measurement [4]. For a likely to survive the post-depositional process than other bones in
forensic anthropologist, the quality of the biological profile the body [11]. The cervical vertebrae exhibit a strong cortical layer
depends on the quantity of recovered skeletal elements and the and the small surface areas expose less bone to taphonomic
elements [9–11]. The dense internal trabecular bone is resilient to
mechanical stresses and the circular vertebral shape increases the
bone’s architectural structural integrity [11–14]. In addition, the
* Corresponding author at: Saint Mary’s University, Department of Anthropology,
cervical vertebrae exhibit morphological characteristics that
923 Robie Street, Halifax, Nova Scotia, B3H 3C3, Canada.
E-mail address: [email protected] (T.R. Peckmann). individualize them from all other skeletal elements, including

http://dx.doi.org/10.1016/j.forsciint.2019.110072
0379-0738/© 2019 Elsevier B.V. All rights reserved.
2 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072

other vertebrae, making them easily identifiable; the unique from the Lopes collection (87 males and 73 females). The Athens
skeletal attributes of cervical vertebrae allow for rapid anatomical collection is comprised of individuals who lived the majority of
sequencing in comparison to other vertebral elements. their lives in the late 20th and early 21st centuries. It is an example
Previous research has demonstrated that the cervical vertebrae of a growing contemporary skeletal reference collection housing
exhibit sexual dimorphism suitable for accurate and reliable sex an estimated 225 individuals. The Athens collection is continu-
estimation [9,12,15–18]. Traditionally, research involving methods of ously accepting unclaimed remains from cemeteries around
sex estimation from cervical vertebrae have focused on three bones: Athens, Greece, and previous research has shown that it provides
atlas (C1), axis (C2), and the transitional seventh vertebra (C7) [9,12,15– the most accurate data available for sex estimation in a
17,19]. The unique morphology of C1, C2, and C7 has created an interest contemporary European population [23]. The Lopes collection is
in studying these bones as compared to the typical third through sixth comprised of historic individuals who lived the majority of their
cervical vertebrae (C3 to C6), which are less often investigated. In lives during the late 19th and early 20th centuries. Unclaimed
addition, previous research has, traditionally, utilized measurements remains are mostly acquired from three major cemeteries within
of the centrum, articular facets, and length and width of the vertebrae the city of Lisbon [22]. Sample selection criteria for the historic
as they have been shown to be highly dimorphic [9,15,17,20,21]. Lopes collection included individuals living the majority of their
However, these characteristics are likely to sustain taphonomic lives after 1900, the year that researchers have observed overall
damage and fragmentation upon recovery from a deposition site [9– positive secular changes (Albanese 2010). Cemetery registrations
11,14,20]. If vertebral preservation is poor, the spinous process and from both collections have resulted in a large range of biographic
features of the transverse processes are the most likely structures to be and demographic data for the individuals studied.
damaged, although the strong architectural construction increases the Three measurements were collected from each of the seven
potential for recovery of the complete vertebral column cervical vertebrae: maximum cervical vertebral body height (CHT),
[9–11,14,16]. Structural rigor make vertebrae ideal for sex estimation cervical anterior-posterior diameter (CAP), and cervical transverse
due to their preservation. The CAP and CTR diameters are enclosed and diameter (CTR); CHT was not collected for the first cervical
protected by the vertebral arches and the CHT is located on the dense vertebra, as this vertebra does not have a centrum. In total, 20
vertebral body, resulting in resiliency to mechanical, taphonomic, and measurements were taken for the purposes of this study. These
architectural stresses [9–11,14]. The CHT, CAP, and CTR morphometric variables are defined in Table 1 and are shown in Figs. 1 and 2. In
characteristics are, therefore, more likely to be present in forensic some instances, it was not possible to collect all three measure-
skeletal cases. Therefore, the current project aims to create a method ments from a single vertebra due to taphonomic damage or mild
for estimating sex from the cervical vertebrae using only the three osteoarthritis. Thus, all statistical analyses were performed using
vertebral measurements most resilient to mechanical, taphonomic, the total number of individuals utilized for each specific
and architectural stresses, and investigating their accuracy for the measurement (i.e. total number of individuals in which CHT,
estimation of sex using all cervical vertebrae, i.e. C1–C7. CAP, and CTR could be examined) rather than the total number of
Overall, while previous research has demonstrated accuracy individuals examined from each skeletal collection as outlined
rates greater than 80% for sex estimation from the cervical above. Measurements were taken using Vernier calipers and
vertebrae, they have cited these methods to be population-specific rounded to the nearest 0.01 mm, following the protocols of
[9,12,15–18]. However, the methods were created and tested on the previous researchers [15,17,18,22,24–29]. For inclusion in this
sample population used in the study and not cross referenced on study, individuals must have at least five of the seven vertebrae
other population samples. Currently, there are no studies that present to anatomically sequence the bones; if one or two
investigate the accuracy of cervical vertebrae for the estimation of vertebrae were missing, sequential ordering and numerical
sex in contemporary Greek and historic Portuguese populations. identification of the cervical bones was possible through anatomi-
Therefore, the aim of this research is to evaluate the relationship cal articulation. Vertebrae exhibiting taphonomic damage, trauma,
between sex and three morphometric characteristics (CHT, CAP, or pathological remodelling were excluded from this study.
and CTR) of the cervical vertebrae in a contemporary Greek and an Statistical analyses were performed using Minitab (version
historic Portuguese population. 17.0) and SPSS (version 21.0) statistical software following the
protocol of Tatarek [18,26]. As with any biological morphometric
2. Materials and methods study, it is important to minimize measurement error and to
understand its influence. Statistical significance levels were,
This research examined 1020 vertebrae utilizing 295 adult therefore, adjusted using a Bonferroni correction in order to
skeletonized individuals (157 males and 138 females) of European reduce the chance of errors associated with multiple comparisons
ancestry between 20 and 99 years of age. Two documented skeletal (Type-1 error). All data were tested for normality and all data were
cemetery collections were utilized: the University of Athens found to be normally distributed. Intra-observer error rates were
human skeletal reference collection (i.e. the Athens collection) in calculated by re-measuring 60 individuals (30 individuals from
Greece, and the Luis Lopes skeletal collection (i.e. the Lopes each collection) one week after the initial measurements were
collection) in Lisbon, Portugal. A total of 295 individuals were recorded. Inter-observer error rates were calculated with the aid of
examined for this research (n=295): 135 individuals from the two research assistants (i.e. one research assistant from the Athens
Athens collection (70 males and 65 females), and 160 individuals skeletal collection and one research assistant from the Lopes

Table 1
Measurement descriptions.

Measurements Descriptiona
Maximum Cervical Vertebral Body The maximum superior to inferior vertebral body height along the anterior border of each vertebra with the exception
Height (CHT) of C1. (Note: C2HT includes the odontoid process).
Cervical Anterior-Posterior The maximum mid-sagittal diameter from the anterior to posterior aspects of the vertebral foramen.
Diameter (CAP)
Cervical Transverse Diameter (CTR) The maximum medio-lateral diameter measured from the left to the right pedicles within the vertebral foramen.
a
Adapted from Clark (1985), Eisentstein (1983), Kibii and colleagues (2010), Taitz (1996), Tatarek (2005), and Verbiest (1955).
 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072 3

differences existed between contemporary (i.e. Athens skeletal

collection) and historic (i.e. Lopes skeletal collection) mean vertebral
measurements as a result of secular change or geographic population.
Results from both collections show that there is no statistically
significant difference between populations (p > 0.002) for CHT, CAP
and CTR. Therefore, the contemporary Athens and historic Lopes
skeletal collections were grouped together into one large indepen-
dent population of “Europeans” for all further statistical analyses.
Discriminant functions were created from this combined
European population. Discriminant functions were created for
each independent cervical vertebra, C1 through C7, using all three
Fig. 1. Anterior view of a typical cervical vertebra depicting the maximum cervical measurements (CHT, CAP, and CTR), to establish whether sex could
vertebral body height (CHT) measurement (Photo credit Andrew S. Rozendaal). be estimated from a single vertebra. Discriminant functions were
also created for each independent cervical vertebra using only
measurements of the vertebral foramen (CAP and CTR) and using
only those vertebral measurements that were most dimorphic
(CTR and CHT). In total, three discriminant functions were created
for each independent cervical vertebra.
In addition, two discriminant functions were created using all seven
cervical vertebrae and all 20 vertebral measurements using both enter
and stepwise methods of regression. These discriminant functions were
generated using 55.6% of the combined sample of Europeans; only a
portion of the individuals (n = 164; 86 males and 78 females) were
utilized because only these individuals had a complete set of seven
undamaged cervical vertebrae. Additional multivariate discrimi-
nant functions were created to investigate whether particular
combinations of vertebrae may prove accurate for sex estimation:
 The first and second vertebrae (C1 and C2) were utilized to create
one discriminant function using all three measurements (CHT,
CAP, and CTR). These vertebrae were examined independently
due to their irregular shape.
 C2–C7 were utilized to create one discriminant function using the
two most dimorphic measurements (CTR and CHT). C1 was
excluded as it does not possess a vertebral body.
 The typical cervical vertebrae (C3 through C6) and the transitional
C7 vertebra were utilized to create three discriminant functions
using all three measurements (CHT, CAP, and CTR), vertebral
foramen measurements only (CAP and CTR), and the two most
Fig. 2. Superior view of a typical cervical vertebra depicting the cervical anterior-
dimorphic measurements (CTR and CHT). These three discrimi-
posterior diameter (CAP), and cervical transverse diameter (CTR) measurements
(Photo credit Andrew S. Rozendaal). nant functions were then recreated after the exclusion of C7.
 The second and fifth vertebrae (C2 and C5) were utilized to create
skeletal collection); thirty-five individuals from the Athens three discriminant functions using all three measurements (CHT,
collection and 29 individuals from the Lopes collection were CAP, and CTR), vertebral foramen measurements only (CAP and
re-measured to test for inter-observer error. These sample sizes are CTR), and the two most dimorphic measurements (CTR and CHT).
appropriate as previous studies have shown that a subsample of These two vertebrae were examined independently since
10%–20% of the total population should be used to test for intra- stepwise discriminant function analysis found them to exhibit
and inter-observer error [30–32]. Both intra-and inter-observer the most sexually dimorphism.
error rates were calculated using paired-sample t-tests for
each measurement variable. In addition, the technical error of Overall, 12 discriminant functions were created using various
measurement (TEM), relative technical error of measurements combinations of cervical vertebrae.
(rTEM), and the coefficient of reliability (R-value) were calculated. To assess accuracy and reproducibility, the discriminant functions
The TEM measures precision by estimating the standard deviation that achieved overall predicted accuracies of 80% and above were
of the differences between two measurement values. The rTEM is cross-validated on an independent sample of 32 individuals from both
the percentage total of the TEM and is used to compare the Athens and Lopes skeletal collections; this is a statistically viable
measurement error across studies using a standard mathematical sample size as cited by other researchers [30–32]. This research used
equation (rTEM = ((total TEM)/mean)  100). Lastly, the R-value is 80% as the minimum requirement because osteological methods
used to indicate the proportion of variance due to factors other utilized for the estimation of sex are generally considered reliable if
than measurement error. they produce accuracies of at least 80% [20,9,33–38].
Descriptive statistics were obtained for each vertebral variable in
the Athens and Lopes skeletal collections, including means and 3. Results
standard deviations, for both males and females (Tables 2 and 3). Two-
sample t-tests were used to determine whether statistically significant 3.1. Intra- and inter-observer error rates
differences existed between male and female mean vertebral
measurements. For males and females, two-sample Table 4 shows the results of intra-and inter-observer error in the
t-tests were also used to examine whether statistically significant Athens collection. An examination of intra-observer error shows no
4 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072

Table 2
Descriptive statistics for the Athens skeletal collection (n = 135), including assessment of sexual dimorphism.

Measurement n Mean (mm) Standard Minimum (mm) Maximum (mm) t-value p-value
Deviation (SD)

C1AP Male 59 31.47 2.02 25.73 37.15 5.91 0.000*

Female 57 29.28 1.97 25.88 33.80
C2AP Male 64 16.22 1.45 12.94 19.86 1.02 0.310
Female 62 15.95 1.50 12.34 19.12
C3AP Male 59 14.13 1.23 11.95 18.11 1.15 0.251
Female 56 13.86 1.23 10.94 16.52
C4AP Male 69 13.54 1.36 10.69 17.43 0.39 0.701
Female 61 13.45 1.28 9.78 16.40
C5AP Male 63 13.63 1.52 10.30 17.47 1.46 0.147
Female 61 13.25 1.41 9.94 16.15
C6AP Male 62 13.49 1.41 10.30 16.72 1.75 0.083
Female 59 13.06 1.27 10.53 16.18
C7AP Male 65 13.97 1.48 10.22 16.67 2.00 0.048
Female 61 13.47 1.34 10.23 15.81

C1TR Male 59 29.24 2.03 25.99 34.47 4.01 0.000*

Female 58 27.81 1.82 23.28 32.67
C2TR Male 64 24.53 1.56 21.41 28.47 4.82 0.000*
Female 62 23.19 1.57 19.93 26.61
C3TR Male 59 23.82 1.28 21.72 27.38 3.48 0.001*
Female 56 22.93 1.46 19.41 25.98
C4TR Male 69 24.68 1.28 22.23 27.69 3.17 0.002*
Female 61 23.92 1.42 20.23 27.14
C5TR Male 63 25.50 1.39 22.98 28.55 3.73 0.000*
Female 61 24.55 1.45 21.72 28.43
C6TR Male 62 25.81 1.29 21.79 28.28 3.80 0.000*
Female 59 24.79 1.64 21.33 29.32
C7TR Male 65 25.01 1.45 21.56 28.96 3.80 0.000*
Female 61 23.93 1.72 19.81 28.20

C2HT Male 65 39.65 3.01 32.57 47.16 9.00 0.000*

Female 62 35.73 1.77 31.96 40.52
C3HT Male 59 13.86 1.38 11.32 17.69 5.83 0.000*
Female 56 12.58 0.96 10.39 14.67
C4HT Male 69 13.80 1.16 11.34 17.51 9.11 0.000*
Female 59 12.11 0.94 10.16 14.71
C5HT Male 62 13.09 1.20 10.03 15.86 6.97 0.000*
Female 60 11.75 0.91 9.88 13.80
C6HT Male 63 13.69 1.11 11.56 16.46 6.94 0.000*
Female 57 12.40 0.92 10.64 15.28
C7HT Male 65 15.43 1.40 11.13 18.14 6.41 0.000*
Female 61 14.01 1.08 11.09 16.21
*
Significant difference at p  0.002.

statistically significant differences between any cervical vertebral 3.2. Assessment of sexual dimorphism
measurements, with the exception of C4TR and C6TR. TEM values are Tables 2 and 3 show the results of the assessment of sexual
low, rTEM values range from 0.18% to 2.60%, and all vertebral dimorphism in the Athens and Lopes skeletal collections,
measurements have an R-value greater than 0.95 with the exception respectively. In the Athens collection, all CTR and CHT measure-
of C4TR (R = 0.94) and C5TR (R = 0.94). An examination of inter- ments demonstrate a statistically significant difference between
observer error shows statistically significant differences in vertebral males and females (p < 0.002). However, for the CAP measurement,
measurements C3AP, C1TR, C3TR, C5TR and C6TR. TEM values are high only the first cervical vertebra (C1) exhibits a statistically
(comparatively), rTEM values range from 4.87% to 15.42%, and all significant difference between males and females. Overall, the
vertebral measurements have an R-value of less than 0.50. CAP measurement does not demonstrate sexual dimorphism in the
Table 5 shows the results of intra-and inter-observer error in Athens collection. Similarly, in the Lopes collection, all CTR and
the Lopes collection. In the Lopes collection, intra-observer CHT measurements demonstrate a statistically significant differ-
error shows no statistically significant differences between ence between males and females (p < 0.002), with the exception of
vertebral measurements, with the exception of C 3 TR, C 5 TR, C1TR and C2TR, and only the first cervical vertebra (C1) exhibits a
C 6 TR, and C4 HT. TEM values are low, rTEM values range from statistically significant difference between males and females for
0.16% to 2.53%, and all vertebral measurements have an R-value the CAP measurement. These results indicate that the CAP
of 0.95 or greater, with the exception of C 3 HT (R = 0.92), C 5 HT measurement does not demonstrate sexual dimorphism in either
(R = 0.93), C6 HT (R = 0.94), C 7 HT (R = 0.94). An examination of the Athens or Lopes skeletal collections. As a result of these
inter-observer error shows no statistically significant differ- findings, males and females have been analysed separately for both
ences in vertebral measurements, with the exception of C1 TR the Athens and Lopes skeletal collections.
and C 3 HT. TEM values are also low, while rTEM values range
from 0.40% to 4.81%, and all vertebral measurements have an R- 3.3. Vertebral variation due to secular change and ancestry
value of an R-value of 0.95 or greater, with the exception of
C 5 AP (R = 0.93), C4 TR (R = 0.94), C 3 HT (R = 0.84), C 4 HT (R = 0.93), Table 6 shows the results of the assessment of secular change
C 5 HT (R = 0.92), and C 6 HT (R = 0.89). and ancestry in the Athens and Lopes collections. Overall, for both
 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072 5

Table 3
Descriptive statistics for the Lopes skeletal collection (n = 160), including assessment of sexual dimorphism.

Measurement n Mean (mm) Standard Minimum (mm) Maximum (mm) t-value p-value
Deviation (SD)

C1AP Male 77 30.61 1.99 25.97 35.09 5.22 0.000*

Female 70 28.92 1.93 24.73 33.61
C2AP Male 82 16.00 1.34 12.58 20.10 2.33 0.021
Female 66 15.52 1.21 12.88 18.40
C3AP Male 81 13.79 1.23 11.14 16.83 1.86 0.065
Female 70 13.44 1.10 10.67 16.22
C4AP Male 83 13.26 1.21 10.51 16.08 1.03 0.306
Female 73 13.06 1.26 10.45 16.68
C5AP Male 80 13.53 1.26 10.59 16.52 2.45 0.015
Female 72 13.02 1.31 10.26 16.51
C6AP Male 84 13.47 1.33 10.71 16.99 2.36 0.020
Female 70 12.99 1.23 9.67 16.09
C7AP Male 81 13.74 1.33 11.01 17.20 2.85 0.005
Female 64 13.12 1.28 10.06 15.73

C1TR Male 77 28.74 2.27 24.02 36.21 2.87 0.005

Female 70 27.75 1.89 24.04 32.67
C2TR Male 84 23.23 1.68 19.94 27.50 2.07 0.040
Female 69 22.70 1.46 18.75 25.18
C3TR Male 80 23.17 1.48 19.61 27.02 3.55 0.001*
Female 71 22.37 1.28 18.81 25.09
C4TR Male 83 24.25 1.60 20.56 27.99 3.95 0.000*
Female 73 23.31 1.36 20.18 27.33
C5TR Male 80 25.04 1.69 20.28 28.71 4.06 0.000*
Female 72 24.05 1.31 20.56 27.65
C6TR Male 84 25.18 1.57 21.24 28.87 4.34 0.000*
Female 70 24.14 1.39 20.48 27.69
C7TR Male 81 24.55 1.69 21.03 28.30 4.72 0.000*
Female 64 23.12 1.28 10.06 15.73

C2HT Male 84 37.73 2.22 33.93 43.76 6.47 0.000*

Female 68 35.63 1.78 31.59 39.04
C3HT Male 80 13.56 1.06 10.64 15.94 7.29 0.000*
Female 69 12.33 1.02 10.32 14.19
C4HT Male 81 13.17 1.13 9.86 15.54 6.87 0.000*
Female 71 11.98 1.00 10.18 14.40
C5HT Male 79 12.76 1.17 10.32 15.07 6.36 0.000*
Female 70 11.66 0.94 9.49 13.51
C6HT Male 83 13.05 1.16 10.47 15.52 5.56 0.000*
Female 68 12.06 1.03 9.84 14.98
C7HT Male 81 14.80 1.16 11.96 17.32 5.56 0.000*
Female 63 13.79 1.02 11.35 16.23
*
Significant difference at p  0.002.

sexes, there are no statistically significant differences between the 3.5. Discriminant functions for combinations of cervical vertebrae
collections (p > 0.002) for CHT, CAP and CTR. Therefore, the two
populations, i.e. Athens and Lopes collections, were combined into All 20 measurements were combined to create one discrimi-
one large independent sample “Europeans” for all statistical nant function to estimate sex using the enter method of regression.
analyses. This function resulted in overall accuracy rates of 84.1% (Function
2; Table 7). When a stepwise method was used, only seven
3.4. Discriminant functions for independent cervical vertebra measurements (C1AP, C2HT, C2TR, C3HT, C5TR, C5HT, C7TR)
exhibited large t-value coefficients, which indicate a high potential
Sex estimation from a single vertebra ranges from 66.9%–74% for for the estimation of sex. The resulting discriminant function
males of the combined European population and from 70.2%–79.5% resulted in an overall accuracy rate of 82.6% (Function 4, Table 7).
for females of the combined European population when all three Additional multivariate discriminant functions were created to
measurements (CHT, CAP, and CTR) are utilized. Discriminant investigate whether particular combinations of cervical vertebrae
functions were also created for each cervical vertebra using only may prove accurate for sex estimation. When only the first and
measurements of the vertebral foramen (CAP and CTR) and using second cervical vertebrae (C1 and C2) were utilized, the overall
only those vertebral measurements that were most sexually accuracy rate was 72.8% using all three measurements (CAP, CTR,
dimorphic (CTR and CHT). When measurements of the vertebral CHT). When C2 through C7 were utilized, the highest accuracy rates
foramen are used (CAP and CTR), analyses demonstrate accuracy were found using the two most dimorphic measurements (CTR and
rates between 61.2% and 70.6% for European males and between CHT). The overall accuracy rate was 84.5%. When the typical
58.7% and 70.1% for European females. When only those vertebral cervical vertebrae (C3 through C6) and the transitional C7 vertebra
measurements that were most sexually dimorphic (CTR and CHT) were utilized, overall accuracy was 83.3% using only the two most
were utilized, accuracy rates increased and ranged from 67.6%–73.3% sexually dimorphic measurements (CTR and CHT). Accuracy rates
for males and 70.4%–78.5% for females. Overall, sex estimation from a decreased when the transitional C7 vertebra was excluded. The
single cervical vertebra is most accurate when all three measure- results of this study indicate that second and fifth cervical
ments (CHT, CAP, an CTR) are utilized. vertebrae (C2 and C5) are the most sexually dimorphic. When
6 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072

Table 4
Results of intra-observer error and inter-observer error in the Athens skeletal collection.

Intra-observer error Inter-observer error

a b c
Measurement n t-value p-value* TEM rTEM R n t-value p-value* TEMa rTEMb Rc
Athens Collection
C1AP 27 1.24 0.225 0.20 0.65 0.99 27 0.09 0.932 2.28 7.55 0.06
C2AP 30 1.46 0.156 0.20 1.20 0.99 34 0.89 0.379 1.25 7.75 0.21
C3AP 29 0.16 0.876 0.15 1.04 0.99 33 4.62 0.000* 1.21 8.54 0.10
C4AP 29 1.75 0.091 0.08 0.58 1.00 33 1.94 0.061 1.59 11.67 0.31
C5AP 29 0.27 0.790 0.14 1.01 0.99 34 0.19 0.853 1.68 12.38 0.26
C6AP 29 0.96 0.344 0.22 1.59 0.97 32 1.26 0.219 1.52 11.41 0.31
C7AP 30 0.27 0.789 0.15 1.01 0.99 35 0.06 0.950 1.49 10.93 0.03
C1TR 27 0.93 0.359 0.06 0.22 1.00 27 5.53 0.000* 1.57 5.53 0.27
C2TR 30 0.51 0.611 0.06 0.26 1.00 34 2.54 0.016 1.41 5.91 0.46
C3TR 29 1.52 0.139 0.10 0.42 1.00 33 3.38 0.002* 1.26 5.44 0.31
C4TR 29 4.32 0.000* 0.06 0.23 1.00 33 2.83 0.008 1.25 5.14 0.31
C5TR 29 1.46 0.156 0.05 0.18 1.00 34 4.79 0.000* 1.22 4.87 0.39
C6TR 29 3.75 0.001* 0.08 0.31 1.00 32 4.44 0.000* 1.60 6.34 0.10
C7TR 30 1.73 0.094 0.19 0.78 0.99 35 2.17 0.037 1.62 6.60 0.22
C2HT 30 1.30 0.205 0.68 0.83 0.98 34 0.77 0.445 3.31 8.81 0.05
C3HT 29 0.61 0.549 0.24 1.79 0.95 33 0.99 0.329 1.86 14.58 0.03
C4HT 29 0.79 0.435 0.34 2.60 0.94 33 0.62 0.541 1.94 15.42 0.06
C5HT 29 0.71 0.482 0.29 2.27 0.94 34 0.01 0.989 1.49 12.31 0.14
C6HT 29 1.37 0.183 0.27 2.07 0.95 32 0.61 0.545 1.60 12.66 0.03
C7HT 30 0.53 0.602 0.26 1.72 0.96 35 0.12 0.905 1.83 12.8 0.06
*
Significant difference at p  0.002.
a
TEM (technical measurement error).
b
rTEM (relative technical measurement error).
c
R (coefficient of reliability).

Table 5
Results of intra-observer error and inter-observer error in the Lopes skeletal collection.

Intra-observer error Inter-observer error

Measurement n t-value p-value* TEMa rTEMb Rc n t-value p-value* TEMa rTEMb Rc

Lopes Collection
C1AP 29 0.53 0.598 0.13 0.45 1.00 29 1.01 0.320 0.19 0.66 0.99
C2AP 30 0.27 0.789 0.07 0.47 0.99 29 1.37 0.180 0.18 1.15 0.98
C3AP 30 0.91 0.368 0.07 0.48 1.00 29 1.14 0.263 0.26 1.85 0.96
C4AP 30 0.64 0.528 0.07 0.53 1.00 28 2.14 0.042 0.23 1.68 0.97
C5AP 30 1.64 0.111 0.11 0.85 0.99 29 0.55 0.586 0.35 2.58 0.93
C6AP 30 0.84 0.407 0.10 0.76 0.99 29 1.23 0.227 0.26 1.93 0.97
C7AP 30 2.99 0.006 0.08 0.58 1.00 27 1.51 0.142 0.29 2.15 0.96
C1TR 29 1.01 0.321 0.19 0.67 1.00 29 16.33 0.000* 0.12 0.40 1.00
C2TR 30 0.35 0.731 0.06 0.28 1.00 29 0.89 0.382 0.11 0.47 1.00
C3TR 30 4.56 0.000* 0.09 0.39 1.00 28 0.35 0.729 0.20 0.89 0.98
C4TR 30 2.20 0.036 0.07 0.28 1.00 28 1.29 0.209 0.32 1.35 0.94
C5TR 30 3.81 0.001* 0.06 0.24 1.00 29 1.74 0.092 0.19 0.76 0.99
C6TR 30 3.83 0.001* 0.04 0.16 1.00 29 2.02 0.053 0.12 0.48 0.99
C7TR 30 2.39 0.024 0.05 0.21 1.00 27 0.52 0.608 0.18 0.76 0.99
C2HT 30 0.77 0.448 0.43 1.18 0.95 28 1.90 0.068 0.43 1.22 0.98
C3HT 30 2.30 0.029 0.35 0.28 0.92 26 3.73 0.001* 0.62 4.81 0.84
C4HT 30 3.98 0.000* 0.25 2.00 0.96 26 2.62 0.015 0.35 2.79 0.93
C5HT 30 1.58 0.126 0.31 2.53 0.93 27 1.25 0.224 0.41 3.33 0.92
C6HT 30 2.16 0.039 0.28 2.22 0.94 27 0.73 0.473 0.45 3.65 0.89
C7HT 30 1.47 0.151 0.29 2.04 0.94 26 1.41 0.170 0.25 1.81 0.98
*
Significant difference at p  0.002.
a
TEM (technical measurement error).
b
rTEM (relative technical measurement error).
c
R (coefficient of reliability).

only the second and fifth cervical vertebrae (C2 and C5) were addition, for the combined European population, the results of this
utilized, overall accuracy rates were 77%. study indicate that a high estimation of sex is achieved when all
Table 7 shows the results of all generated discriminant cervical vertebrae (C1 through C7) and all measurements (CAP, CTR,
functions that achieved an overall accuracy rate of 80% or higher. CHT) are utilized.
The results of this study demonstrate that combinations of
vertebrae provide a more accurate estimate of sex than single or 3.6. Biases in sex estimation
independent cervical vertebrae; this indicates that the accuracy of
sex estimation is enhanced when the most sexually dimorphic Table 7 shows that correct discriminant function classification
measurements (CTR and CHT) from all cervical vertebrae are by sex is greater in females in all functions, apart from Function 1,
utilized (excluding C1 due to an absence of the CHT variable). In whereas males are classified more correctly than females. The
 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072 7

Table 6
Results of the assessment of secular change/ancestry in the Athens and Lopes collections.

Males Females

Measurement Athens (n) Lopes (n) t-value p-value Athens (n) Lopes (n) t-value p-value
C1AP 59 77 2.47 0.015 57 70 1.03 0.305
C2AP 64 82 0.94 0.349 62 66 1.82 0.071
C3AP 59 81 1.59 0.115 56 70 2.00 0.048
C4AP 69 83 1.30 0.194 61 73 1.76 0.080
C5AP 63 80 0.41 0.682 61 72 0.95 0.342
C6AP 62 84 0.07 0.944 59 70 0.34 0.734
C7AP 65 81 0.97 0.336 61 64 1.47 0.144
C1TR 59 77 1.35 0.180 58 70 0.19 0.852
C2TR 64 84 4.90 0.000* 62 69 1.84 0.068
C3TR 59 80 2.78 0.006 56 71 2.25 0.026
C4TR 69 83 1.85 0.067 61 73 2.53 0.013
C5TR 63 80 1.76 0.080 61 72 2.04 0.043
C6TR 62 84 2.67 0.008 59 70 2.39 0.018
C7TR 65 81 1.77 0.079 61 64 2.22 0.029
C2HT 65 84 4.32 0.000* 62 68 0.32 0.746
C3HT 59 80 1.33 0.188 56 69 1.38 0.171
C4HT 69 81 3.39 0.001* 59 71 0.79 0.431
C5HT 62 79 1.65 0.100 60 70 0.56 0.578
C6HT 63 83 3.36 0.001* 57 68 1.94 0.055
C7HT 65 81 2.92 0.004 61 63 1.16 0.247
*
Significant difference at p-value  0.002.

Table 7
Discriminant function equations (that achieved an accuracy rate of 80% or higher) for combinations of cervical vertebrae in the combined European population.

Function Discriminant function equation Sectioning Eigenvalue Canonical Correct Overall

point* correlation classification correct
by sex classification

% Males % Females n %
1 y = 0.240(C2TR) + 0.197(C2HT) + 0.179(C3TR) + 0.327(C3HT) 0.232(C4TR) + 0.0625 0.809 0.669 85.0 83.0 158/ 84.5
0.251(C4HT) 0.322(C5TR) + 0.153(C5HT) + 0.234(C6TR) + 0.101(C6HT) + 0.358 187
(C7TR) 0.018(C7HT) 17.246
2 y = 0.201(C1AP) + 0.015(C1TR) 0.111(C2AP) 0.384(C2TR) + 0.151(C2HT) + 0.0485 1.004 0.708 83.7 84.6 138/ 84.1
0.222(C3AP) + 0.177(C3TR) + 0.325(C3HT) 0.447(C4AP) 0.180(C4TR) + 0.135 164
(C4HT) + 0.252(C5AP) 0.467(C5TR) + 0.338(C5HT) 0.105(C6AP) + 0.133(C6TR)
+ 0.068(C6HT) + 0.058(C7AP) + 0.508(C7TR) 0.058(C7HT) 15.219
3 y = 0.003(C3TR) + 0.377(C3HT) 0.130(C4TR) + 0.342(C4HT) 0.367(C5TR) + 0.05 0.692 0.639 82.9 83.9 165/ 83.3
0.107(C5HT) + 0.215(C6TR) + 0.104(C6HT) + 0.395(C7TR) + 0.133(C7HT) 16.568 198
4 y = 0.190(C1AP) 0.355(C2TR) + 0.175(C2HT) + 0.363(C3HT) 0.430(C5TR) + 0.0465 0.917 0.692 77.3 88.2 157/ 82.6
0.428(C5HT) + 0.565(C7TR) 16.994 190
5 y = 0.170(C3AP) + 0.012(C3TR) + 0.389(C3HT) 0.487(C4AP) 0.169(C4TR) + 0.052 0.747 0.654 81.9 82.8 163/ 82.3
0.375(C4HT) + 0.158(C5AP) 0.335(C5TR) + 0.108(C5HT) + 0.120(C6AP) + 0.189 198
(C6TR) + 0.037(C6HT) 0.005(C7AP) + 0.409(C7TR) + 0.119(C7HT) 15.536
6 y = 0.061(C3AP) + 0.103(C3TR) + 0.327(C3HT) 0.372(C4AP) 0.118(C4TR) + 0.0555 0.646 0.627 80.2 81.2 175/217 80.6
0.374(C4HT) + 0.138(C5AP) 0.271(C5TR) + 0.101(C5HT) + 0.209(C6AP) + 0.412
(C6TR) + 0.196(C6HT) 16.478
7 y = 0.089(C3TR) + 0.306(C3HT) 0.064(C4TR) + 0.373(C4HT) 0.367(C5TR) + 0.049 0.584 0.607 80.2 80.4 175/ 80.3
0.092(C5HT) + 0.498(C6TR) + 0.251(C6HT) 16.974 218
*
Females < Sectioning point value < Males.

variation between the correct classification of males and females is validation test, indicating that they are strong statistical algo-
within 2%. However, Function 4 utilizes the step-wise regression rithms. Functions 1 and 6 resulted in cross-validation accuracies
formula, in which the variation between male and female accuracy lower than 80%, indicating weaker algorithms for sex estimation.
rates is 10.9%, 77.3% and 88.2% respectively. Thus, functions 1 and 6 are not recommended for forensic
anthropological application.
3.7. Cross-validation
4. Discussion
For the Athens and Lopes skeletal collections, the seven
discriminant functions that achieved overall accuracy rates of 4.1. Intra- and inter-observer error rates
80% or greater were cross-validated on an independent sample of
32 individuals. This sample size is appropriate as previous studies The methods and techniques used in forensic anthropology
have shown that approximately 10%–20% of the total population must show high levels of reliability, accuracy, and precision [39].
size is required for testing an independent sample [30,31]. The Accuracy assesses the degree of correctness and, for estimation of
results of cross-validation are shown in Table 8. Functions 2, 3, 4, 5 sex in forensic anthropology, methods must show accuracy rates of
and 7 achieved accuracy rates greater than 80% in the cross- 80% or greater to be acceptable [5,9,33–40]. Precision is the ability
8 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072

Table 8
Cross-validation classification accuracies for the combined European population (based on Table 7, discriminant function equations that achieved an accuracy rate of 80% or
higher).

Function Discriminant function equation Total n (M/F) Cross-validation correct classification

n %
1 y = 0.240(C2TR) + 0.197(C2HT) + 0.179(C3TR) + 0.327(C3HT) 0.232(C4TR) + 0.251(C4HT) 23 (13/10) 18/23 78.3
0.322(C5TR) + 0.153(C5HT) + 0.234(C6TR) + 0.101(C6HT) + 0.358(C7TR) 0.018(C7HT) 17.246
2 y = 0.201(C1AP) + 0.015(C1TR) 0.111(C2AP) 0.384(C2TR) + 0.151(C2HT) + 0.222(C3AP) + 0.177 19 (10/9) 16/19 84.2
(C3TR) + 0.325(C3HT) 0.447(C4AP) 0.180(C4TR) + 0.135(C4HT) + 0.252(C5AP) 0.467(C5TR) +
0.338(C5HT) 0.105(C6AP) + 0.133(C6TR) + 0.068(C6HT) + 0.058(C7AP) + 0.508(C7TR) 0.058
(C7HT) 15.219
3 y = 0.003(C3TR) + 0.377(C3HT) 0.130(C4TR) + 0.342(C4HT) 0.367(C5TR) + 0.107(C5HT) + 23 (10/13) 19/23 82.6
0.215(C6TR) + 0.104(C6HT) + 0.395(C7TR) + 0.133(C7HT) 16.568
4 y = 0.190(C1AP) 0.355(C2TR) + 0.175(C2HT) + 0.363(C3HT) 0.430(C5TR) + 0.428(C5HT) + 0.565 23 (9/14) 20/23 87.0
(C7TR) 16.994
5 y = 0.170(C3AP) + 0.012(C3TR) + 0.389(C3HT) 0.487(C4AP) 0.169(C4TR) + 0.375(C4HT) + 0.158 23 (11/12) 19/23 82.6
(C5AP) 0.335(C5TR) + 0.108(C5HT) + 0.120(C6AP) + 0.189(C6TR) + 0.037(C6HT) 0.005(C7AP) +
0.409(C7TR) + 0.119(C7HT) 15.536
6 y = 0.061(C3AP) + 0.103(C3TR) + 0.327(C3HT) 0.372(C4AP) 0.118(C4TR) + 0.374(C4HT) + 0.138 24 (9/15) 19/24 79.2
(C5AP) 0.271(C5TR) + 0.101(C5HT) + 0.209(C6AP) + 0.412(C6TR) + 0.196(C6HT) 16.478
7 y = 0.089(C3TR) + 0.306(C3HT) 0.064(C4TR) + 0.373(C4HT) 0.367(C5TR) + 0.092(C5HT) + 24 (9/15) 21/24 87.5
0.498(C6TR) + 0.251(C6HT) 16.974

for a method to consistently produce repeatable results and, for vertebra. However, with the exception of the first cervical vertebra
the estimation of sex in forensic anthropology, must show less than (C1), CAP diameters did not exhibit statistically significant sexual
10% error to be accepted [5,9,33–40]. Accuracy rates and precision dimorphism. The etiology of the C1AP dimorphism may be attributed
levels that fall within the acceptable standards determine the to sexual variation between the male and female cranium [9]. Studies
reliability of the method. In forensic anthropological research, by Holland [41] and Gapert and colleagues [42] have shown that the
accuracy and precision error rates must be presented to demon- cranial base (i.e. occipital condyles and foramen magnum) exhibits
strate that the techniques utilized for the estimation of sex are sexual dimorphism. The C1 cradles the weight of the skull and
created using scientifically accepted principles that produce consequently shares a functional relationship with the cranial base.
results that are statistically greater than chance [39,38]. The dimorphic structures of the cranial base will, therefore, influence
Intra- and inter-observer error tests measure the precision levels the morphological structure of C1, such as the articular facets and the
of measurement reproducibility. In the Athens collection, the vertebral foramen [9,12].
evaluation of intra- and inter-observer error demonstrated error Predictive development models of vertebral growth by previous
variations that were less than 10% (Table 4). Error variations that are researchers have also shown that, of the three currently studied
less than 10% indicate acceptable precision standards [9,20,33–37]. characteristics (CAP, CTR, CHT), the CTR and CHT measurements
However, the results of inter-observer error (i.e. TEM, rTEM, and develop more slowly and for a longer period of time than CAP and
R) indicate issues with replicability of vertebral measurements. are therefore influenced by secondary sexual development and
Given the large raw value measurement discrepancies, this is environmental factors [24,26,51]. The results of vertebral growth
likely the result of misunderstanding or misinterpretation by the indicate that, by six years of age, the vertebral foramen has reached
research assistant as English was not their first language. In the approximately 90% of its complete adult size. During this
Luis Lopes collection, the evaluation of intra- and inter-observer developmental time, biomechanical forces will change the
error also demonstrated error variations that were less than 10%, vertebral foramen from a circular to a triangular shape to reflect
with the exception of C1TR (Table 4). However, the inter- the necessary range of neck motions that will morphologically
observer error for C1TR exhibited a 25.18% error (Table 5). This protect the spinal cord [24]. The majority of dimensional increases
may have also resulted from a misinterpretation of the C1TR occur in the CTR diameter, which reaches its adult size by
definition by the research assistant as English was not their approximately 10 years of age [24,26,51]. Therefore, the develop-
first language. Overall, the CTR variable exhibits the least ment of CTR extends into early puberty. The height of the vertebral
consistency with measurement repeatability as compared to bodies (CHT) has the longest period of growth reaching its full
CAP and CHT. Imprecision is likely decreased if the morpho- length at approximately 20 years of age [26,52,53], which may
metrics are performed by trained individuals. Therefore, experi- attribute to its greater sexual dimorphism compared to the
ence in morphometrics is essential and the authors suggest extra vertebral foramen measurements (CAP, CTR).
care and training when measuring and explaining the CTR Overall, the results of the current study are consistent with the
measurement variable, both in research and practical contexts. majority of previous research, which has also found minimal
Future research should focus on clarification and standardization dimorphism in the CAP measurement [18,24,43–46]. However,
of measurements of the cervical vertebrae. some studies have demonstrated conflicting results, noting the
existence of dimorphism in CAP diameters among various
4.2. Assessment of sexual dimorphism populations [9,15,17,21,25,26,42]. Human morphometrics can vary
between populations due to differences in ancestral groups,
The results of the current research indicate that the CHT genetic distance, environmental factors, and socioeconomic status,
measurement is the most dimorphic variable of the cervical which can influence the size and shape of skeletal characteristics
vertebrae, followed by the CTR measurement. These results are [9,22,24,25,47]. These influences may account for the differences
consistent with previous research that has also found CHT and CTR to displayed between the current research study and other popula-
be more dimorphic within other populations [9,15–17,19,24,25]. In tion studies that examined cervical vertebrae.
both the Athens and Lopes skeletal collections, mean CAP diameter In addition, variations in the methodological approaches and
values were larger in males than in females for every cervical statistical tools applied by the researchers in each study may be
 A.S. Rozendaal et al. / Forensic Science International 306 (2020) 110072 9

related to the non-biological variability [48]. Sex estimation explore population-specificity of these discriminant functions
methods accept a type-1 error rate of 5% to identify whether using larger sample sizes.
relationships exist between measurements when a p-value  0.05 The European discriminant function equations achieved
is used to evaluate statistical significance [48–50]. Type-1 error is accuracy rates up to 84.5%. The results of this study indicate that
the misclassification of a relationship between testable variables, the accuracy of sex estimation is enhanced when all measurements
which is not biologically present, caused by procedural multiplicity from all cervical vertebrae are utilized; the more variables utilized
error rather than a quantitative error in the data set. When a in sex estimation methods from the cervical vertebrae, the more
p-value of 0.05 is selected, a ‘false positive’ is likely to occur five accurate the result. Therefore, when available, all measurements
times out of every 100 tests when identifying variable relation- and all cervical vertebrae should be utilized, as they are better
ships; Type-1 error results in a ‘false positive’ when examining predictors of sex in the European population. While the cervical
relationships between variables. To reduce the chances of a’ false vertebrae have proved useful for the estimation of sex from
positive’, a Bonferroni correction adjusts the significance level, skeletonized remains, the discriminant functions derived from the
since there is a greater likelihood that an error appears in the present study should be utilized only in cases in which other, more
statistical outcome as the number of testable variables increases. In accurate predictors of sex (i.e. the skull and the pelvis) are not
the current research, this adjustment made statistical analyses available for analyses.
more rigorous in identifying statistical significance.
CRediT authorship contribution statement
4.3. Discriminant functions for cervical vertebrae
Andrew S. Rozendaal: Conceptualization, Data curation,
A single vertebra did not accurately estimate sex at, or above, the
Formal analysis, Methodology, Project administration, Resources,
80% accuracy threshold. Studies have shown that with every increase
Software, Validation, Visualization, Writing - original draft,
in the number of measurements used in a discriminant function, the
Writing - review & editing. Shelby Scott: Formal analysis,
accuracy for estimating the sex also increases [15,16]. This is
Validation, Writing - original draft, Writing - review & editing.
consistent with the current research, which has found that utilizing a
Tanya R. Peckmann: Supervision, Writing - review & editing.
greater number of measurements and a greater number of cervical
Susan Meek: Writing - review & editing.
vertebrae achieves higher accuracies than utilizing fewer measure-
ments and fewer bones. For instance, when two cervical vertebrae
(C1 and C2) and all three measurements (CHT, CAP, and CTR) were Acknowledgments
used to estimate sex, the function achieved an overall accuracy rate of
72.8%. When the number of cervical vertebrae increased to four Special thanks are extended to Saint Mary’s University for
(C3–C6), the accuracy of the discriminant function utilizing all three awarding this research the Canadian Governor General’s Gold
measurements (CHT, CAP, and CTR) increased to 80.6%. When all Academic Medal for 2016. Thanks are extended to the University of
seven cervical vertebrae (C1 to C7) and all three measurements (CHT, Athens, Greece, Dr. Sortiris Manolis and Dr. Constantine Moun-
CAP, and CTR) were used, the discriminant function achieved an even trakis for allowing access to their skeletal collection. Also, thanks to
higher accuracy rate of 84.1% in the combined European population. the National Museum of Natural History and Science, Portugal, and
This is consistent with the findings of other researchers; with every Dr. Susana Garcia for allowing access to their skeletal collection.
increase in the number of measurements used in a discriminant Thanks are extended to Maria-Eleni Chovalopoulou and Natasha
function, the accuracy for estimating the sex also increases [15,16]. Carvalho for volunteering as research assistants in Greece and
Only seven discriminant functions, from the current study, Portugal, respectively.
achieved predictive accuracies at, or above, 80%. A cross-validation
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