World number five and the tournament's top seed, Aryna Sabalenka, was upset by American Amanda Anisimova 3-6 6-4 6-3 in the third round at the Charleston Open.
Sabalenka's serve was the key to her early success, connecting on 73 per cent of her first serves, and winning 15 of the 19 points in that situation (79 per cent).
When Sabalenka's first serve percentage fell to 48 in the second set, Anisimova found her footing, breaking twice in the frame, before pouncing on the Belarusian's lacklustre second serve in the deciding set, winning seven of nine opportunities to earn the break and the win.
Sixth seed Jessica Pegula was also bounced in surprising fashion, losing 6-4 3-6 6-4 to unseeded compatriot Coco Vandeweghe.
In a razor-close contest, it came down to who took their chances, as Vandeweghe saved seven of the 10 break points she faced, while Pegula could only save one of five.
World number 10 Ons Jabeur had no problem navigating the challenge of Emma Navarro, winning 6-3 6-2 as she broke her opponent's serve on six occasions.
Canadian seventh seed Leylah Hernandez also fell victim to an unseeded upset, going down 3-6 6-3 6-4 against Poland's Magda Linette.
In a meeting between two top-10 seeds, Belinda Bencic got the better of Madison Keys to edge it 6-4 6-4.
Second seed Paula Badosa had to come from a set down to eventually get the better of Claire Liu 3-6 7-6 (10-8) 6-1.
In the late session, French 12th seed Alize Cornet went down in a close tussle against Ukraine's Anhelina Kalinina 7-6 (7-5) 7-5, while Jabeur won her second match of the day, beating Irina Begu 6-3 6-2.
Meanwhile, in Bogota at the Copa Colsanitas, top seed Camila Osorio dispatched Turkish opponent Ipek Oz 6-3 7-6 (7-4) to move on to the quarter-final where she will meet Elina Avanesyan.