Joseph and Jennifer Cartellone conceived their daughter Rebecca, 24, through in-vitro fertilisation in 1994 after fertility struggles.
They are now suing a hospital and fertility clinic claiming another man's sperm was used to create the embryo and Rebecca's biological father may be a doctor from the hospital.
Rebecca gave her parents DNA test kits to trace their family tree as a gift last Christmas, but when the results came back two months later they showed that she didn't share any of her father's genetic history and makeup.
A separate paternity test confirmed Mr Cartellone was not his daughter's biological father.
Mr and Mrs Cartellone, from the US city of Delaware, Ohio, are suing the Institute for Reproductive Health, The Christ Hospital and Ovation Fertility, all of Cincinnati.
Lawyers hired by the family claim the search for Rebecca's biological father has been narrowed down to five men.
One was a doctor at the Christ Hospital when she was conceived, they claim.
Mr and Mrs Cartellone are seeking unspecified damages as they sue for negligence and breach of contact.
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