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Williams, the seven-time Wimbledon champion, slipped and hurt her heavily-strapped right leg before quitting her first round tie after just six games against Aliaksandra Sasnovich.
She used her racket to help her stand, but only so she could limp to the net to concede, just the second mid-match retirement at any Grand Slam tournament of her career and first since 1998.
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Williams' latest bid for a record-tying 24th Grand Slam title ended with her "heartbroken" and in tears after just 30 minutes and less than a set.
The 39-year-old was leading 3-1 in the first set of her first round match against Aliaksandra Sasnovich of Belarus when she slipped and injured her right leg.
Serena, a seven-time Wimbledon champion, is still one shy of Margaret Court's Grand Slam singles record of 24.
Serena Williams has previously won four Olympic gold medals, three of them in women's doubles partnering with her sister Venus.
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The 39-year-old American went down 6-3, 7-5 at Roland Garros to the Kazakh 21st seed, leaving her still one short of Margaret Court's all-time record for most Grand Slam singles titles.
World number one Djokovic, chasing a 19th Grand Slam title, faces 19-year-old Italian Lorenzo Musetti who is in his first Grand Slam tournament.
Williams employed verbal motivation to help her recover from a 4-1 deficit in the second set of a 6-4, 6-4 victory over fellow American Danielle Collins in the third round of the French Open on Friday
Russian second seed Daniil Medvedev and Alexander Zverev are also in action, while Victoria Azarenka will be looking to extend her best Roland Garros run since 2015.
The 39-year-old Williams, who has been one short of Margaret Court's all-time record of most major trophies since 2017, brushed off a second-set blip to win 6-3, 5-7, 6-1.
Williams made her comments in a post-match news conference following her first-round victory Monday at the French Open.
The 39-year-old Williams is again looking to equal Margaret Court's all-time record of 24 Grand Slam singles titles, but arrives in Paris having lost two of her three matches on clay.