French Open champion Garbine Muguruza lost her first match since winning her debut grand slam at the Mallorca Open on Tuesday.
Muguruza was beaten 6-3 6-4 by Kirsten Flipkens in the opening round of the tournament - making its debut on the WTA Tour - the Belgian taking just an 74 minutes.
The new world number two struggled for rhythm on her return after Roland Garros success, serving five double faults and failing to save a single break point against Flipkens.
But Muguruza - who shocked Serena Williams in the French Open final - made a strong start with a break in the opening game of the match, however her advantage was soon wiped out.
Flipkens reeled off three successive games and continued to attack the top seed's serve, while also saving five of the six break points she faced to line up a meeting against Monica Puig, who beat Julia Goerges in straight sets.
Second seed Jelena Jankovic is safely through to the next round after beating Ana Konjuh 7-5 6-3, her next opponent is qualifier Elise Mertens after she overcame Francesca Schiavone in three sets.
Sabine Lisicki, Wimbledon finalist in 2013, is also through after beating fourth seed Kristina Mladenovic 6-4 6-4. The German finally setting up a clash with Mariana Duque-Marino after letting five match points slip through her fingers.
Fifth seed Yulia Putintseva also bowed out of the tournament at the hands of Sorana Cirstea, while Anastasija Sevastova overcame Stefanie Voegele.
Next up for Sevastova is Eugenie Bouchard, the Canadian getting back to winning ways with a 6-3 7-5 victory over world number 53 Danka Kovinic - who gave Petra Kvitova a scare at Roland Garros.
There were also victories for Veronica Cepede, Laura Siegemund and Anna-Lena Friedsam.