Sex estimation of the adult Neandertal Regourdou 1 (Montignac, France): Implications for sexing human fossil remains.

Sex is a biological trait fundamental to the study of hominin fossils. Among the many questions that can be addressed are those related to taxonomy, biological variability, sexual dimorphism, paleoobstetrics, funerary selection, and paleodemography. While new methodologies such as paleogenomics or paleoproteomics can be used to determine sex, they have not been systematically applied to Pleistocene human remains due to their destructive nature. Therefore, we estimated sex from the coxal bone of the newly discovered pelvic remains of the Regourdou 1 Neandertal (Southwest France, MIS 5) based on morphological and metric data employing two methods that have been recently revised and shown to be reliable in multiple studies. Both methods calculate posterior probabilities of the estimate. The right coxal bone of Regourdou 1 was partially reconstructed providing additional traits for sex estimation. These methods were cross validated on 14 sufficiently preserved coxal bones of specimens from the Neandertal lineage. Our results show that the Regourdou 1 individual, whose postcranial skeleton is not robust, is a male, and that previous sex attributions of comparative Neandertal specimens are largely in agreement with those obtained here. Our results encourage additional morphological research of fossil hominins in order to develop a set of methods that are applicable, reliable, and reproducible.

The influence of biological relatedness on sexual dimorphism and sex classification based on external morphology of the frontal bone.

The most significant sexual differences in the human skull are located in the upper third of the face (the frontal bone), which is a useful research object, mainly in combination with virtual anthropology methods. However, the influence of biological relatedness on sexual dimorphism and frontal bone variability remains unknown. This study was directed at sexual difference description and sex classification using the form and shape of the external surface of the frontal bones from a genealogically documented Central European osteological sample (nineteenth to twentieth centuries). The study sample consisted of 47 cranial CT images of the adult members of several branches of one family group over 4 generations. Three-dimensional virtual models of the frontal bones were analyzed using geometric morphometrics and multidimensional statistics. Almost the entire external frontal surface was significantly different between males and females, especially in form. Significant differences were also found between this related sample and an unrelated one. Sex estimation of the biologically related individuals was performed using the classification models developed on a sample of unrelated individuals from the recent Czech population (Cechová et al. in Int J Legal Med 133: 1285 1294, 2019), with a result of 74.46% and 63.83% in form and shape, respectively. Failure of this classifier was caused by the existence of typical traits found in the biologically related sample different from the usual manifestation of sexual dimorphism. This can be explained as due to the increased degree of similarity and the reduction of variability in biologically related individuals. The results show the importance of testing previously published methods on genealogical data.

Classification performance of the Sella-Tunis et al. (2017) sex estimation method in Czech population: different posterior probability threshold approaches.

In this study we tested classification performance of a sex estimation method from the mandible originally developed by Sella-Tunis et al. (2017) on a heterogeneous Israeli population. Mandibular linear dimensions were measured on 60 CT scans derived from the Czech living population. Classification performance of Israeli discriminant functions (DFs-IL) was analyzed in comparison with calculated Czech discriminant functions (DFs-CZ) while different posterior probability thresholds (currently discussed in the forensic literature) were employed. Our results comprehensively illustrate sensitivity of different discriminant functions to population differences in body size and degree of sexual dimorphism. We demonstrate that the error rate may be biased when presented per posterior probability threshold. DF-IL 1 showed least sensitivity to population origin and fulfilled criteria of sufficient classification performance when applied on the Czech sample with a minimum posterior probability threshold of 0.88 reaching overall accuracy ≥ 95%, zero sex bias, and 80% of classified individuals. The last parameter was higher in DF-CZ 1 which was the main difference between those two DFs suggesting relatively low dependance on population origin. As the use of population-specific methods is often prevented by complicated assessment of population origin, DF-IL 1 is a candidate for a sufficiently robust method that could be reliably applied outside the reference sample, and thus, its classification performance deserves further testing on more population samples.

Prioritizing a high posterior probability threshold leading to low error rate over high classification accuracy: the validity of MorphoPASSE software for cranial morphological sex estimation in a contemporary population.

An increasing number of software tools can be used in forensic anthropology to estimate a biological profile, but further studies in other populations are required for more robust validation. The present study aimed to evaluate the validity of MorphoPASSE software for sex estimation from sexually dimorphic cranial traits recorded on 3D CT models (n = 180) from three populations samples (Czech, French, and Egyptian). Two independent observers performed scoring of 4 cranial traits (2 of them bilateral) in each population sample of 30 males and 30 females. The accuracy of sex estimation using traditional posterior probability threshold (pp = 0.5) ranged from 85.6% to 88.3% and overall classification error from 14.4% to 11.7% for both observers, and corresponds to the previously published values of the method. The MorphoPASSE method is also affected by the subjectivity of the observers, as both observers show agreement in sex assignment in 83.9% of cases, regardless of the accuracy of the estimates. Applying a higher posterior probability threshold (pp 0.95) provided classification accuracy of 97.9% and 93.3% of individuals (for observer A and B respectively), minimizing the risk of error to 2.1% and 6.7%, respectively. However, sex estimation can only be applied to 54% and 66% of individuals, respectively. Our results demonstrate the validity of the MorphoPASSE software for cranial sex estimation outside the reference population. However, the achieved classification success is accompanied by a high risk of errors, the reduction of which is only possible by increasing the posterior probability threshold.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

The Importance of Historical Medical Records Review in the Interpretation of Dry Bone Lesions on Identified Skeletal Remains: A Case Study of a Polymorbid Male (1895-1940).

ABSTRACT: The examination of documented skeletal remains provides an exceptional opportunity for biohistorical research to answer questions about an individual's life and death. Research in this area also makes it possible to assess the reliability of historical records from the period of interest, which is often the subject of discussion, especially in cases of historically known individuals. The remains of K.B.C. (1895-1940), a prominent local landowner and politician, were exhumed because of the repair of a family tomb in Jíloviste, Czech Republic. The aim of this study was to analyze pathological changes in his bones and to interpret these by comparing them with the results of a historical medical records review of private family and public archives regarding his diseases and death, thus verifying their credibility. Morphological and X-ray examinations of the bones revealed several serious pathological changes, whose presence fully corresponded to the studied documents. This showed the records' reliability, and it was thus possible to accurately interpret the lesions found. The results demonstrated the need for interdisciplinary collaboration in the analysis of such cases, including the assistance of the living descendants of the studied individuals, if possible.

Familial occurrence of skeletal developmental anomalies as a reflection of biological relationships in a genealogically documented Central European sample (19th to 20th centuries).

Skeletal developmental anomalies (SDA) are a subject of constant interest across scientific disciplines, but still mostly as isolates and curiosities. The aim of this study was to find out to what extent the occurrence of SDA reflects documented biological relationships. The skeletal remains of 34 individuals with known genealogical data were available, members of one family over four generations (19th to 20th centuries, Bohemia, Czech Republic), including some inbred individuals. The occurrence of 89 SDA was assessed on the basis of scopic morphological evaluation and X-ray and CT examinations. The degree of similarity between individuals was calculated using a "similarity coefficient" (SC). A linear model was used to test the relationship between positive values of the SC and the relatedness of biologically related individuals. Simultaneously, based on population frequencies of the evaluated anomalies, those that could be considered familial were recorded. A statistically significant relationship between morphological similarity and the biological distance between individuals was found. The greatest similarity was found among close relatives such as parents and children, siblings, or grandparents and grandchildren. The effect of increased consanguinity on the occurrence of anomalies was not confirmed, however. Seventeen SDA shared by closely related individuals were found in the sample, supporting the documented family relationships among them. Eleven of these were selected as possibly familial, but only five were statistically significant: an elongated styloid process, a cervical block vertebrae (arch, facet joints), hamate hamulus aplasia, anteater nose sign, and incomplete fusion of the S1 spinous process. There were also 28 cases of individual occurrences of 17 different SDA, without connection to the documented relationships between individuals.

Age-related differences in cranial sexual dimorphism in contemporary Europe.

Biomechanical load and hormonal levels tended to change just like the soft and skeletal tissue of the elderly with age. Although aging in both sexes shared common traits, it was assumed that there would be a reduction of sexual dimorphism in aged individuals. The main goals of this study were (1) to evaluate age-related differences in cranial sexual dimorphism during senescence, (2) to determine age-related differences in female and male skulls separately, and (3) to compare skull senescence in Czech and French adult samples as discussed by Musilová et al. (Forensic Sci Int 269:70-77, 2016). The cranial surface was analyzed using coherent point drift-dense correspondence analysis. The study sample consisted of 245 CT scans of heads from recent Czech (83 males and 59 females) and French (52 males and 51 females) individuals. Virtual scans in the age range from 18 to 92 years were analyzed using geometric morphometrics. The cranial form was significantly greater in males in all age categories. After size normalization, sexual dimorphism of the frontal, occipital, and zygomatic regions tended to diminish in the elderly. Its development during aging was caused by morphological changes in both female and male skulls but secular changes must also be taken into account. The most notable aging changes were the widening of the neurocranium and the retrusion of the face, including the forehead, especially after the age of 60 in both sexes. Sexual dimorphism was similar between the Czech and French samples but its age-related differences were partially different because of the population specificity. Cranial senescence was found to degrade the accuracy of sex classification (92-94%) in the range of 2-3%.

Biological relationships and frontal sinus similarity in skeletal remains with known genealogical data.

Frontal sinus analysis has potential utility for detecting biologically related individuals. However, the methodological approach to its evaluation, as well as its informative value, have been questioned. The aim of this work is to introduce a new approach to evaluating the frontal sinus using the 'external supraorbital line' (ESOL) and to determine whether there are sex differences within families in frontal sinus measurements and whether frontal sinus similarity reflects known genetic relationships in both measurements and morphology. We examined the skeletal remains of 41 adult individuals (25 males, 16 females), all members of one family over four generations (19th to 20th centuries), including individuals with very close consanguinity. CT images of skulls were acquired, and both the dimensions and morphology of the frontal sinuses were analyzed using their portions above the ESOL. No significant sex differences were found within families based on frontal sinus dimensions. Significant relationships were found between biological distance and the maximum height and morphology of the frontal sinuses. The greatest degree of similarity was found among closely related individuals. Additionally, in several cases, there was a greater degree of similarity between first cousins or grandparents and their grandchildren than among siblings or parents and their children. Total surface, volume and width are not significant indicators of relatedness. Known genetic relationships are also supported by individual morphological features. Variability within families with very close consanguineous relationships was lower than within families with common degrees of consanguinity, although differences are significant only for some variables.

Advanced procedures for skull sex estimation using sexually dimorphic morphometric features.

This paper introduces an automated method for estimating sex from cranial sex diagnostic traits by extracting and evaluating specialized morphometric features from the glabella, the supraorbital ridge, the occipital protuberance, and the mastoid process. The proposed method was developed and evaluated using two European population samples, a Czech sample comprising 170 crania reconstructed from anonymized CT scans and a Greek sample of 156 crania from the Athens Collection. It is based on a fully automatic algorithm applied on 3D models for extracting sex diagnostic morphometric features which are further processed by computer vision and machine learning algorithms. Classification accuracy was evaluated in a population specific and a population generic 2-way cross-validation scheme. Population-specific accuracy for individual morphometric features ranged from 78.5 to 96.7%, whereas population generic correct classification ranged from 71.7 to 90.8%. Combining all sex diagnostic traits in multi-feature sex estimation yielded correct classification performance in excess of 91% for the entire sample, whereas the sex of about three fourths of the sample could be determined with 100% accuracy according to posterior probability estimates. The proposed method provides an efficient and reliable way to estimate sex from cranial remains, and it offers significant advantages over existing methods. The proposed method can be readily implemented with the skullanalyzer computer program and the estimate_sex.m GNU Octave function, which are freely available under a suitable license.

A case of marked bilateral asymmetry in the sacral alae of the Neandertal specimen Regourdou 1 (Périgord, France).

OBJECTIVES: A marked asymmetry was previously reported in the sacral alae and S1-L5 facets orientation of the Neandertal individual Regourdou 1. Here, we provide a detailed description and quantification of the morphology and degree of asymmetry of this sacrum. MATERIAL AND METHODS: Regourdou 1 was compared to a modern human sample composed of 24 females and 17 males, and to other Neandertal individuals. Both traditional and geometric morphometric analyses were used in order to quantify the degree of sacral asymmetry of Regourdou 1. RESULTS: The asymmetry of both sacral alae and facets orientation substantially exceeds directional and absolute asymmetry of the healthy modern sample. Regourdou 1 shows a considerably shorter right ala, which is absolutely and relatively outside of the modern and Neandertal variations. CONCLUSION: Regourdou 1 shows marked sacral asymmetry that probably originated in early ontogenetic development. An asymmetric sacrum reflects asymmetric load dissipation and could relate to other morphological abnormalities observed in the skeleton, especially the mild scoliosis of the spine and the asymmetry of the femoral diaphyses. Further investigation is necessary to elucidate the relationship between those morphologies as well as a potential impact on the life of the individual.

A validation study of the Stoyanova et al. method (2017) for age-at-death estimation quantifying the 3D pubic symphyseal surface of adult males of European populations.

The age-at-death estimation thresholds have recently been shifted towards a more objective assessment of the aging process. Such a non-subjective approach offers quantitative methods of age estimation; for instance, the method relating to the surfaces of pubic symphyses of males published by Stoyanova et al. (J Forensic Sci 62:1434-1444, 2017). A validation study was conducted to test the method performance in European samples. The sample consisted of 96 meshes of pubic symphyses of male individuals (known sex and age) that came from four different samples (two Portuguese collections, one Swiss, and one Crete). Stoyanova's method based on five regression models (three univariate and two multivariate models) performed worse in our sample, but only when the whole sample (without age limitation) was included. A sample limited to individuals under 40 years of age achieved better results in our study. The best results were reached through the thin plate spline algorithm (TPS/BE) with a root mean square error of 5.93 years and inaccuracy of 4.47 years. Generally, the multivariate regression models did not contribute to better age estimation. In our sample in all age categories, age was systematically underestimated. The quantitative method tested in this study works best for individuals under 40 years of age and provides a suitable basis for further research.

Sex estimation using external morphology of the frontal bone and frontal sinuses in a contemporary Czech population.

Sex estimation is a task of utmost importance in forensic anthropology and bioarcheology. Along with the pelvic bone, the skull is the most important source of sexual dimorphism. On the human skull, the upper third of the face (i.e., the frontal bone) is one of the most significant sexually dimorphic structures useful in anthropological research, especially when studied by methods of virtual anthropology. This study was focused on sex estimation using the form and shape of the external surface of the frontal bone with or without the inclusion of its sinuses. The study sample consisted of 103 cranial CT images from a contemporary Czech population. Three-dimensional virtual models of the frontal bones and sinuses were analyzed using geometric morphometrics and multidimensional statistics: coherent point drift-dense correspondence analysis (CPD-DCA), principal component analysis (PCA), and support vector machine (SVM). The whole external frontal surface was significantly different between males and females both in form and shape. The greatest total success rate of sex estimation based on form was 93.2%, which decreased to 86.41% after crossvalidation, and this model identified females and males with the same accuracy. The best estimation based on shape reached a success rate of 91.26%, with slightly greater accuracy for females. After crossvalidation, however, the success rate decreased to 83.49%. The differences between sexes were significant also in the volume and surface of the frontal sinuses, but the sex estimation had only 64.07% accuracy after crossvalidation. Simultaneous use of the shape of the frontal surface and the frontal sinuses improved the total success rate to 98.05%, which decreased to 84.46% after crossvalidation.

A method of sexing the human os coxae based on logistic regressions and Bruzek's nonmetric traits.

OBJECTIVES: This study aims at proposing a visual method for sexing the human os coxae based on a statistical approach, using a scoring system of traits described by Bruzek (2002). This method is evaluated on a meta-population sample, where the data were acquired by direct observation of dry bones as well as computed tomography (CT) scans. A comparison with the original Bruzek's (2002) method is performed. MATERIALS AND METHODS: Five hundred and ninety two ossa coxae of modern humans are included in the reference dataset. Two other samples, composed respectively of 518 ossa coxae and 99 CT-scan images, are both used for validation purposes. The individuals come from five European or North American population samples. Eleven trichotomic traits (expressing female, male, or intermediate forms) were observed on each os coxae. The new approach employs statistical processing based on logistic regressions. An R package freely available online, PELVIS, implements both methods. RESULTS: Both methods provide highly reliable sex estimates. The new statistical method has a slightly better accuracy rate (99.2%) than the former method (98.2%) but has also a higher rate of indeterminate individuals (12.9% vs. 3% for complete bones). CONCLUSION: The efficiency of both methods is compared. Low error rates were preferred over high ability of reaching the classification threshold. The impact of lateralization and the asymmetry of observed traits are discussed. Finally, it is shown that this visual method of sex estimation is reliable and easy to use through the graphical user interface of the R package.

Architecture of the femoral and tibial diaphyses in relation to body mass and composition: Research from whole-body CT scans of adult humans.

OBJECTIVES: Recent investigations have evaluated the influence of body composition on long bones in order to overcome the limits of body mass (BM) estimation methods and eventually lead to studying nutrition in past populations. Knowing how fat mass (FM) and fat-free mass (FFM) impact the skeleton would also enhance the understanding of mobility, activity, and locomotion derived from bone architecture. We investigated the relationship between BM and composition, and the architecture of the entire tibial and femoral diaphyses in an adult sample representative of a wide range of variation in age, BM, and composition. MATERIALS AND METHODS: Body composition was measured directly from 78 whole-body CT scans for which the age, sex, BM, and stature were recorded. The entire diaphyseal thickness, volume, curvature, and cross-sectional geometry parameters of both the femur and tibia were numerically extracted. RESULTS: FM correlates with large portions of the femoral thickness in females only. FFM correlates with the femoral diaphysis in males but not in females. FFM correlates with the tibia architecture in both sexes, while FM is correlated in males exclusively. DISCUSSION: BM and body components influence the architecture of the diaphysis of lower limb long bones in sex-specific patterns that are mostly reflected in their thickness and can be recorded, in some cases, for their strength, rigidity, and volume. Our results suggest that (1) long bone diaphyses should be thoroughly studied, as a whole, when possible; and (2) BM and body components should be accounted for when deriving activity, mobility, or locomotion patterns from cortical bone.

Kinship and morphological similarity in the skeletal remains of individuals with known genealogical data (Bohemia, 19th to 20th centuries): A new methodological approach.

OBJECTIVES: This article proposes a new approach, called the "similarity coefficient" (SC) for verifying family relationships from skeletal remains using nonmetric traits. Based on this method and further analyses, the authors aim to show the degree of similarity between individuals with varying degrees of kinship, including inbred individuals. MATERIALS AND METHODS: Our sample includes the skeletal remains of 34 individuals with known genealogical data (four generations, 19th to 20th centuries). A total of 243 skeletal nonmetric traits were evaluated with respect to their anatomical characteristics. The SC was calculated by quantifying the agreement of trait occurrence between individuals. We also identified the traits that support the biological relationships of particular individuals by accounting for their population frequencies. RESULTS: There was a positive correlation between the morphological similarity of biologically related individuals and their biological distance. In some cases, we found greater degree of morphological similarity between first cousins than among other close relatives such as parents and children. At the same time, there was no statistically significant difference in the degree of similarity between inbred individuals and common relatives. Proven family relationships were best reflected by cranial traits, especially bone bridges associated with the courses of blood vessels and nerves. CONCLUSIONS: The use of skeletal nonmetric traits for the detection of relatives is possible. There is a relationship between biological distance and the degree of morphological similarity in related individuals. It also appears that inbreeding, despite previous assumptions, does not lead to a significant reduction in morphological variation.

Sex-specific functional adaptation of the femoral diaphysis to body composition.

OBJECTIVES: The human femoral diaphysis is often used to reconstruct loading histories (mobility, activity, body mass). The proximal femur is known to be differentially affected by changes in total fat-mass (FM), fat-free mass (FFM), and body fat percentage (BF%), but the adaptation of the entire diaphysis to body composition has not been thoroughly characterized to date. Understanding how the femoral diaphysis adapts to body components would benefit biomechanical interpretations of the femoral variation and nutrition-related studies. METHODS: Combining various methods from clinical nutrition, biological anthropology, and geometric morphometrics, we evaluated the correlation of measures taken on the entire femoral diaphysis with estimated FM, FFM, and BF% from 61 CT scans (17 females, 44 males). The sample was predominantly composed of people with obesity. RESULTS: Cortical area of the cross-sections and local cortical thickness showed high correlation with BF% in particular, in females only. The curvature significantly decreased with FM and BF% in both sexes. The lowest correlations are found with FFM. CONCLUSIONS: The observed sexual dimorphism is consistent with differing aging processes; cortical bone decreases in females through endosteal resorption while it remains almost constant in males who compensate for endosteal resorption by periosteal apposition on the diaphyseal surface. The functional adaptation to compressive forces indicates a systemic endosteal apposition of bone material with increased BF% and FM in females only. FM and BF% are linked to a straighter femur in both sexes, suggesting an optimization of the resistance to compressive loads by distributing them more linearly along the entire diaphysis.

Disregarding population specificity: its influence on the sex assessment methods from the tibia.

Forensic anthropology has developed classification techniques for sex estimation of unknown skeletal remains, for example population-specific discriminant function analyses. These methods were designed for populations that lived mostly in the late nineteenth and twentieth centuries. Their level of reliability or misclassification is important for practical use in today's forensic practice; it is, however, unknown. We addressed the question of what the likelihood of errors would be if population specificity of discriminant functions of the tibia were disregarded. Moreover, five classification functions in a Czech sample were proposed (accuracies 82.1-87.5 %, sex bias ranged from -1.3 to -5.4 %). We measured ten variables traditionally used for sex assessment of the tibia on a sample of 30 male and 26 female models from recent Czech population. To estimate the classification accuracy and error (misclassification) rates ignoring population specificity, we selected published classification functions of tibia for the Portuguese, south European, and the North American populations. These functions were applied on the dimensions of the Czech population. Comparing the classification success of the reference and the tested Czech sample showed that females from Czech population were significantly overestimated and mostly misclassified as males. Overall accuracy of sex assessment significantly decreased (53.6-69.7 %), sex bias -29.4-100 %, which is most probably caused by secular trend and the generally high variability of body size. Results indicate that the discriminant functions, developed for skeletal series representing geographically and chronologically diverse populations, are not applicable in current forensic investigations. Finally, implications and recommendations for future research are discussed.

Semiautomatic extraction of cortical thickness and diaphyseal curvature from CT scans.

The understanding of locomotor patterns, activity schemes, and biological variations has been enhanced by the study of the geometrical properties and cortical bone thickness of the long bones measured using CT scan cross-sections. With the development of scanning procedures, the internal architecture of the long bones can be explored along the entire diaphysis. Recently, several methods that map cortical thickness along the whole femoral diaphysis have been developed. Precise homology is vital for statistical examination of the data; however, the repeatability of these methods is unknown and some do not account for the curvature of the bones. We have designed a semiautomatic workflow that improves the morphometric analysis of cortical thickness, including robust data acquisition with minimal user interaction and considering the bone curvature. The proposed algorithm also performs automatic landmark refinement and rigid registration on the extracted morphometric maps of the cortical thickness. Because our algorithm automatically reslices the diaphysis into 100 cross-sections along the medial axis and uses an adaptive thresholding method, it is usable on CT scans that contain soft tissues as well as on bones that have not been oriented specifically prior to scanning. Our approach exhibits considerable robustness to error in user-supplied landmarks, suppresses distortion caused by the curvature of the bones, and calculates the curvature of the medial axis.

Validation and reliability of the sex estimation of the human os coxae using freely available DSP2 software for bioarchaeology and forensic anthropology.

OBJECTIVES: A new tool for skeletal sex estimation based on measurements of the human os coxae is presented using skeletons from a metapopulation of identified adult individuals from twelve independent population samples. For reliable sex estimation, a posterior probability greater than 0.95 was considered to be the classification threshold: below this value, estimates are considered indeterminate. By providing free software, we aim to develop an even more disseminated method for sex estimation. MATERIALS AND METHODS: Ten metric variables collected from 2,040 ossa coxa of adult subjects of known sex were recorded between 1986 and 2002 (reference sample). To test both the validity and reliability, a target sample consisting of two series of adult ossa coxa of known sex (n = 623) was used. The DSP2 software (Diagnose Sexuelle Probabiliste v2) is based on Linear Discriminant Analysis, and the posterior probabilities are calculated using an R script. RESULTS: For the reference sample, any combination of four dimensions provides a correct sex estimate in at least 99% of cases. The percentage of individuals for whom sex can be estimated depends on the number of dimensions; for all ten variables it is higher than 90%. Those results are confirmed in the target sample. DISCUSSION: Our posterior probability threshold of 0.95 for sex estimate corresponds to the traditional sectioning point used in osteological studies. DSP2 software is replacing the former version that should not be used anymore. DSP2 is a robust and reliable technique for sexing adult os coxae, and is also user friendly.