Ay
Egyptologist Bob Brier suggested that Ay murdered Tutankhamun in order to usurp the throne, a claim which was based on X-ray examinations of the body done in 1968. He also alleged that Ankhesenamun and the Hittite prince she was about to marry were also murdered at his orders. This murder theory was not accepted by all scholars, and more detailed CT scans of the mummy undertaken by National Geographic (published in late 2005) suggested that Tutankhamun did not die from a blow to his head as Brier had theorized. The "National Geographic" forensic researchers instead presented a new theory that Tutankhamun died from an infection caused by a badly broken leg since he is often portrayed as walking with a cane due to spina bifida, a hereditary trait in his family on his father's side. The bone fragments found in Tutankhamun's skull were most likely the result of post-mortem damage caused by Howard Carter's initial examination of the boy king "because they show no evidence of being inundated with the embalming fluid used to preserve the pharaoh for the afterlife". However, Brier has stated that the bone fragment in the skull is not relevant to the issue of whether Tutankhamun was murdered, acknowledging that it was likely caused by the embalmers. The evidence Brier presents for the murder is a dark spot on the base of the skull, indicating a blow to the head. Dr. Gerald Irwin agrees with Brier on this point.
Ankhesenamun
The two fetuses found buried with Tutankhamun have been proven to be his children, and the current theory is Ankhesenamun is their mother. Not enough DNA was able to be retrieved from the mummies in KV21 to make positive identities of the queens. Enough DNA was pulled to show that the mummy known as KV21a fits as the mother of the two fetuses in Tutankhamun's tomb. The assumption that she is Ankhesenamun fit with her being the only known wife of Tutankhamun in the historical record.