Serena Williams’ preparations for the French Open were dented by another early exit on clay, with the top seed losing to Katerina Siniakova in Parma.
The American was beaten 7-6 (7-4) 6-2 by the Czech world number 68 in round two of the Emilia-Romagna Open.
Williams had accepted a wildcard into the tournament after defeat in her opener at the Italian Open last week.
The 39-year-old has less than two weeks to sharpen her game on clay before the start of the French Open on May 30.
She is not scheduled to compete in any other events before Roland Garros, where she will be chasing a record-equalling 24th Grand Slam singles title.
The American has lost the last three Slam finals she has contested as she aims to draw level with Australian Margaret Court’s milestone.
She could seek a late wildcard into next week’s Strasbourg event, as she did with this Parma tournament, to get in some more match practice.
In her opener on Monday, Williams had comfortably beaten teenager Lisa Pigato but the 25-year-old Siniakova was a trickier prospect.
The Czech former world number one doubles player edged the American in a first-set tie-break before winning 15 of the last 17 points in the second set.