Testing the sex of ancient individuals from their enamel proteins

3 minute read

A recent paper by Nicolas Stewart and colleagues presents a way to determine the sex of ancient individuals by examining the composition of their tooth enamel: “Sex determination of human remains from peptides in tooth enamel”.

Amelogenin is a protein component of tooth enamel. Humans have two different genes for amelogenin, AMELX on the X chromosome, and AMELY on the Y chromosome. The protein products of these genes have slightly different amino acid sequences. Enamel from males, who have both X and Y chromosomes, has a mix of the two proteins, while females have only the AMELX product.

Stewart and coworkers etch the ancient enamel with an acid, freeing protein fragments, or peptides. They use a mass spectrometer to detect the signatures of the Y chromosome and X chromosome specific peptide variations.

The ability to determine the sex of infant and juvenile remains completely revolutionizes studies of growth, child care, epidemiology, and demography in the past. For the first time, it will allow osteologists to examine sex-specific cultural treatment and differentiate between the health of boys and girls, as well as sex-specific growth trajectories and past developmental milestones, such as age of puberty and subsequent repercussions for fertility. Sites with poor preservation are common in archaeological contexts, and at such sites teeth generally survive better than bone, and thus sex can be established for adults as well as juvenile skeletons in the absence of key skeletal identifiers. In addition, the dimorphic peptide sequence is identical in apes (Fig. S1) and so should be present in all hominins.

This is potentially very important for a number of questions that cannot be answered from archaeological sites without direct evidence of sex. The sex of young children in particular is almost impossible to establish reliably from skeletal indicators. In some recent contexts, sex can be inferred from grave goods or from inscriptions. But for most ancient people–including Neandertals and other Paleolithic populations–the sex of young children is unknown.

Amelogenin has previously been used as a forensic sex determination test in humans and in other mammal species. In humans, this test fails to identify males a small fraction of the time because the Y chromosome sometimes has large deletions that include the Y amelogenin (AMELY) gene. That implies that a small fraction of misidentification (biased toward misidentifying males as females) should result from this test applied to ancient samples.

It’s not known whether this difference between males and females has any functional consequences for enamel. Some researchers have been interested in whether the presence of an additional amelogenin variant in males might influence caries. In some populations, females are more likely to have caries than males. The AMELY protein makes up only around 10 percent of the total amelogenin in a tooth, as the Y chromosome version of the gene is not expressed as highly as the X version, but the presence of an alternative form of the protein might make some difference. However, it is very hard to test whether sex-specific life history traits, such as diet differences during development, pregnancy, or other environmental factors, might instead lead to a difference in caries risk.

At any rate, caries incidence was very, very low during most of our evolutionary history compared to the past 20,000 years (and is low in nearly all other mammals). The amelogenin in both sexes did its job pretty well for most of our existence.


Stewart, N. A., Gerlach, R. F., Gowland, R. L., Gron, K. J., & Montgomery, J. (2017). Sex determination of human remains from peptides in tooth enamel. Proceedings of the National Academy of Sciences, 114(52), 13649-13654.