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.