Venus, Osaka exit Cincinnati in first round
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Williams, 42, who won her last match at the Midwestern pre-US Open event in 2019, lost to 2016 champion Karolina Pliskova 7-5, 6-1.
Osaka's summer form slump deepened meanwhile as the four-time Grand Slam winner was defeated 6-4, 7-5 by China's Zhang Shuai.
Pliskova, one of the game's biggest servers, struck eight aces and broke four times in a win over Williams lasting for nearly one and three-quarter hours.
Williams, semi-finalist from a decade ago, has gone winless in her last five matches, last earning a victory in the Wimbledon first round.
Pliskova said she had to lift her level to run away with the second set.
"Venus is a great champion, she's a fighter with a big game and big serve," the Czech said.
"She played well at certain points in the first set, I'm feeling physically strong so I was able to up my game in the second set."
Osaka's first-round defeat was the second in as many weeks for Japan's former world number one, now ranked 39th.
The 24-year-old was eliminated in the Toronto first round and has won just a single match out of four on cement -- at the WTA Silicon Valley in July.
World number 44 Zhang moved into the second round in 75 minutes as she won her first main draw match here since 2014.
The Chinese player was on the winning Cincinnati doubles team in 2021 and claimed US Open doubles honours in 2021 with Australian Sam Stosur.
The 33-year-old Zhang was broken while serving for the match as Osaka pulled to five-all; a subsequent break of the crowd favourite and a hold to love by Zhang concluded the victory.
"It was a really good win for me," Zhang said. "At the start I was not feeling my serve but at the baseline I played very well.
"I tried to focus on myself today.
"It's always tough to pay a really good friend. Naomi is amazing but she didn't have the best day today."
The win brings Zhang level at 3-3 in her series with Osaka.