Serena Williams, playing the 1,000th Tour-level match of her glittering career, suffered a shock 7-6(6) 7-5 defeat by Argentina's Nadia Podoroska in the the Italian Open second round on Wednesday.
Playing her first competitive match in nearly three months, the 23-times Grand Slam champion twice came back from a break down but struggled on her first serve as Podoroska took the opening set in the tiebreak.
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Podoroska, who reached the French Open semi-finals last year, produced a string of heavy topspin forehands to take control of the match with an early break in the second set.
Although Podoroska was broken to love on her first attempt to serve out the match, she maintained her composure to break again and secure a place in the round of 16.
Defeat for Williams, who received an opening round bye in Rome, was a major blow to her preparations for the French Open, starting on May 30.
It was the American's 149th defeat in her 1,000 career WTA matches.
World number two Naomi Osaka also crashed out of the tournament, losing 7-6(2) 6-2 to American Jessica Pegula.
Osaka, a four-times Grand Slam winner, has often struggled on clay and her defeat in Rome followed a second-round loss at the Madrid Open this month.
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