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Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM(ox)IRPPY peptide (monoisotopic [M + 2 H]+2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called ‘Lovers of Modena’, two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the ‘Lovers of Modena’ (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

Evidence of maternal care and childbirth events in the past are rare in the archaeological record and are difficult to recognize. To combat this, we analyzed thirteen double burials potentially related to childbirth death events, thereby containing an adult and a perinate. The specimens were excavated from the archaeological area identified as “Forli Campus” (Forli, Italy), that dated to 17th-18th centuries AD and was adjacent to a hospital in use at that time. This period witnessed the development of medical techniques and novel approaches in obstetrics in Europe, with the introduction of lying-in hospitals and maternity wards. We here tested if the double burials were ascribable to childbirth death events and thus represent the first reported cases of the hospitalization of childbirth in the history of medicine. A multidisciplinary analysis was undertaken to achieve this aim, combining anthropology, archaeology, paleopathology and archaeogenetics. In five burials the adult individual was recognized as a female in fertile age and the non-adult individual was assigned as perinate. Mitochondrial DNA analysis highlighted different haplotypes among the individuals of these burials, and these results, combined with the archaeological and anthropological data do not support a possible maternal relationship between them. This study is novel in testing the hypotheses of childbirth deaths, through a reliable approach in the interpretation of these archaeological contexts. The analysis of ancient DNA in this particular application proves a useful strategy to support and complete the interpretation of archaeological and anthropological data, showing that a general assumption of mother/child relations within such burials can be misleading.

In 2014 an archaeological area dated between the 17th and the 18th centuries and located in the historical downtown of the city of Forlì (FC), in Northern Italy, offered the possibility to retrieve and study a huge amount of burials, some of which contained perinatal individuals buried in the same grave with adults. The location of this archaeological site (identified as “Forlì Campus”), near the area of the ancient city hospital, generated the idea that this site could have been the hospital cemetery in the 18th century. The possibility to study the bisome burials, represented by an adult and a perinatal individual, coupled with the period to which they date, make this sample of particularly interest. In fact, since prehistoric times, pregnancy, labor, birth and obstetrics have been women’s field.