*world Number 2, Osaka, in second consecutive clay defeat
Serena Williams saw the 1,000th match of her illustrious career end in a straight-set defeat by Argentina’s Nadia Podoroska at the Italian Open.
The 39-year-old American, playing her first competitive match since mid-February, was beaten 7-6 (8-6) 7-5.
Podoroska looked assured in her first meeting with the 23-time Grand Slam champion as she reached the last 16.
Japanese world number two Naomi Osaka suffered her second successive defeat on clay by losing to Jessica Pegula.
Meanwhile, Australian world number one Ashleigh Barty reached the third round by beating Kazakhstan’s Yaroslava Shvedova 6-4 6-1.
On a day where nine of the women’s top 10 were playing in Rome, most of the attention was focused on the return of Williams to match action.
The American’s last appearance came in the Australian Open semi-final defeat by Osaka on 18 February.
Being pitted against 44th-ranked Podoroska, who surprisingly reached the Roland Garros semi-finals last year, looked to be far from ideal in her bid to spend time on court in the Italian capital this week.
Eighth seed Williams lacked intensity and struggled on first serve throughout the match, landing just 48% of them, and was unable to take any of the three break opportunities which came her way.
Podoroska initially struggled to close out the match as Williams wiped out the Argentine’s 5-2 lead for 5-5 in the second set, before Podoroska regained her composure to beat a top-10 opponent for the third time in her career.
Williams is not scheduled to play again before the French Open starts on 30 May but she hinted after this defeat that she could now look to add a tournament to her schedule.
“It was definitely kind of good to go the distance and to try to be out there, but clearly I can do legions better,” she told a news conference.
“Maybe I do need a few more matches, so I’m going to try to figure that out with my coach and my team and see what we would like to do.
“I have been training for months, but it feels definitely different on clay to make that last adjustment.”
Osaka, who received a first-round bye, lost 7-6 (7-2) 6-2 to American world number 31 Pegula in Rome.
The four-time Grand Slam champion, 23, has often struggled on clay and negative body language – including when she smashed a racquet – showed her discomfort against a confident Pegula.
After a tight opener where she missed three set points, Osaka’s level dropped in the second and Pegula took control.
Pegula, 27, kept her focus and discipline to take advantage of Osaka’s increasingly wayward hitting, breaking for 4-2 when Osaka walloped a wild volley wide and going on to seal the biggest win of her career.
For Osaka, this defeat followed a second-round loss at the Madrid Open earlier this month and comes two and a half weeks before the start of the French Open.