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Roma were handed a 3-0 Italian Cup defeat to Spezia on Friday after they made six substitutions during a chaotic last 16 clash they had already lost earlier in the week.
Paulo Fonseca's side were eliminated on Tuesday after a 4-2 extra-time defeat at the Stadio Olimpico, which they finished with nine-men after initially recovering from an early two-goal deficit to take the match beyond 90 minutes.
The capital city club were down to nine less than two minutes into extra time after Gianluca Mancini and goalkeeper Pau Lopez were both dismissed, and in the aftermath of the second red card brought on Daniel Fuzato and Roger Ibanez to take their total of subs over the allowed maximum of five.
In a statement Serie A, which organises the Italian Cup, said the 3-0 loss has been awarded because it was a larger margin of defeat than the one handed to Roma on the field.
Daniele Verde and Riccardo Saponara compounded Roma's misery in extra-time, days after a humiliating 3-0 thumping at the hands of local rivals Lazio.
The substitution error led to Roma, who had suffered another 3-0 loss at the start of the league season for fielding an unregistered player against Hellas Verona, sacking team manager Gianluca Gombar.
Roma now have to take on Spezia again at the weekend as Fonseca tries to calm a crisis in the Eternal City which has reportedly threatened his job.
On Friday, Fonseca said he had the backing of the team and club but reports in Italy claim there was a brawl in the Roma dressing room in the aftermath of the Spezia defeat, and tensions are high between the coach and captain Edin Dzeko.
Dzeko and midfielder Lorenzo Pellegrini also reportedly demanded the reinstatement of Gombar, who was popular among the players.
Roma are fourth in Serie A, level on 34 points with third-placed Napoli and nine behind league leaders AC Milan ahead of Saturday's match.