England captain Phil Vickery and centre Mike Tindall have been ruled out of Saturday's crucial RBS Six Nations clash with Wales in Cardiff.
Tindall scored England's second try against France
Prop Vickery is still suffering from concussion, while Tindall is in hospital with a knee infection.
England, featuring 11 changes from the team that lost to Ireland, beat France 26-18 at Twickenham on Sunday.
"It's unfortunate for Mike and England because I felt he played a key part in our success," said Ashton.
The coach, who is set to name his starting XV on Tuesday, faces a dilemma at fly-half after impressive performances from Toby Flood and replacement Shane Geraghty in the absence of the injured Jonny Wilkinson.
Wilkinson is still a doubt with the strained hamstring that kept him out of the Ireland game, while Flood will be available to train later in the week after suffering a dead leg against France.
Wales are going to be hurting after the Italy game and we can expect a fair bit of a backlash down there
The 35-year-old Mike Catt should retain the captaincy in Vickery's absence though a problem with his left hamstring is being assessed by the England medical team.
Catt was recalled in place of injured centre Andy Farrell, who will not join up with the squad but remain with Saracens to continue the rehabilitation of his lower back.
England's players will assemble at Bath University on Monday to begin preparing for the Cardiff clash that could ensure a best Six Nations campaign since 2003.
England are level on points at the top of the table with France and Ireland, and victory over Wales could even see Ashton's side crowned champions.
But with a vastly inferior points difference, England would realistically need Italy to beat Ireland in Rome and Scotland to defeat France in Paris.
"We saw glimpses of the sort of rugby we could play but you've got to be consistent to be at the top of the world game," said Ashton.
"I will be looking for another good performance next Saturday and further on down the line leading to the World Cup."
Open-side flanker Tom Rees, the Twickenham man-of-the-match, added: "Wales are going to be hurting after the Italy game [Wales lost 23-20 in controversial fashion] and we can expect a fair bit of a backlash down there."