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.
