Maria Sharapova pulled out with a thigh injury while leading her second-round tie in Rome against Mirjana Lucic-Baroni, just hours after she was denied a wild card for the French Open on Tuesday.
Sharapova, who was wearing a bandage on her left thigh, retired from her contest against the veteran Croat while ahead 4-6, 6-3, 2-1 to round out a miserable day for the five-time Grand Slam champion.
Defeat in Rome meant the 30-year-old also missed out on a chance to qualify for the main draw at Wimbledon, which she could have achieved by reaching the finals in the Italian capital.
While Sharapova will at least have a chance to qualify for Wimbledon, the French Open will be without the twice former winner as well as Roger Federer and the pregnant Serena Williams.
"You can get a wild card when you return from injury but you cannot get a wild card when returning from a doping suspension," French tennis federation president Bernard Giudicelli said in an internet media conference.
"I appreciate the media impact of Maria, I appreciate the broadcasters` expectations but in conscience, it was not possible to go beyond the anti-doping code and beyond the application of the rules.
"I`m very sorry for Maria, very sorry for her fans," added Giudicelli. "They might be disappointed, she might be very disappointed, but it`s my responsibility, my mission, to protect the high standards of the game."
Four-time Rome men`s champion Novak Djokovic overcame Britain`s Aljaz Bedene 7-6 (7/2), 6-2 for a place in the third round.
The world number two came through a tight opening set by dominating the tiebreaker before finishing off his victory amid a smattering of jeers from the crowd.
The Serb lost to Rafael Nadal in last week`s Madrid semi-finals, but his quick start on Tuesday was just the tonic for Djokovic, who has never failed to reach the last eight in Rome.
"It was a little bit of a slower start, Bedene is the kind of player that gives you good rhythm. I had some good exchanges, some good games with rallies and it felt right, especially in the second set," Djokovic said.
"I played a great tiebreak, served very well when I needed to. I just wish that I had started a little bit sharper. But hopefully the next one will be good."
British fifth seed Johanna Konta reached the third round with a crushing 6-3, 6-0 win over Yulia Putintseva. She was joined by double Grand Slam winner Svetlana Kuznetsova as the seventh seed beat Katerina Siniakova 6-1, 7-6 (7/3).
Australian Daria Gavrilova beat former finalist and 10th seed Madison Keys 2-6, 7-5, 7-5, while Dutch 15th seed Kiki Bertens put out Monica Niculescu of Romania 2-6, 6-2, 6-1.