Top seeds Sloane Stephens and Madison Keys suffered shock first-round losses at the Citi Open, while Coco Gauff also departed on Tuesday.
Stephens, the 2015 champion and top seed, went down to Swede Rebecca Peterson 6-2 7-5 at the WTA International event in Washington.
Peterson managed to break Stephens' serve five times on her way to an upset win over the world number eight.
Keys was also stunned, losing to 17-year-old wildcard Hailey Baptiste, who was playing her first WTA Tour main-draw match and claimed a 7-6 (7-4) 6-2 win.
In main-draw action for the first time since her incredible run to the last 16 at Wimbledon, 15-year-old Gauff bowed out with a 6-4 6-2 loss to Zarina Diyas.
While Stephens and Keys were upset, two other seeds – Hsieh Su-wei and Monica Puig – encountered no problems.
Hsieh brushed past Kirsten Flipkens 6-4 6-3 and Puig was too good for wildcard Allie Kiick 6-4 6-2.
Kristina Mladenovic beat Shelby Rogers 6-4 6-4, wildcard Catherine Mcnally overcame Zhu Lin 6-4 6-2 and Camila Giorgi got past Sachia Vickery 6-2 7-5.
Venus' struggles continue, Azarenka eases through at Silicon Valley Classic
Venus Williams' tough run of form continued with a first-round loss to Bethanie Mattek-Sands at the Silicon Valley Classic, while Victoria Azarenka cruised through.
Williams has won just two matches since the French Open and the American veteran went down to Mattek-Sands 6-7 (4-7) 6-3 6-1 in a battle of the wildcards at the WTA Premier tournament in San Jose, California on Tuesday.
The seven-time grand slam singles champion had won her previous four matches against Mattek-Sands, but that run came to an end.
Azarenka, meanwhile, is continuing to enjoy a fine season and was untroubled by qualifier Harmony Tan, winning 6-2 6-4.
The two-time Australian Open champion set up a last-16 clash with Donna Vekic, the Croatian fifth seed having overcome Misaki Doi 6-4 3-6 6-4.
Vekic was one of two seeds in action, with Danielle Collins also progressing, getting past Zhang Shuai 6-4 5-7 6-2.
Meanwhile, Zheng Saisai hammered Heather Watson 6-3 6-1 and Madison Brengle breezed past Andrea Petkovic 6-0 6-3.
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