Team USA suffered a shocking loss when Venus, left, and Serena Williams lost in the first round of doubles. (Martin Bernetti/AFP/Getty Images)

There won’t be a fourth Olympic gold medal in doubles for the Williams sisters.

By Sunday night, Team USA had competed in 12 tennis matches at the Rio Olympics — four in men’s singles, four in women’s singles, two in women’s doubles and two in men’s doubles.

Of those 12 matches, the U.S. has lost in half — none of which were more shocking than top-seeded Serena Williams and Venus Williams’s 6-3, 6-4 loss to the Czech Republic duo of Lucie Safarova and Barbora Strycova in the first round.

It was the first doubles loss in the Olympics for the Williams sisters, who had won Olympic gold in 2000, 2008 and 2012, and the loss is just the latest setback for Team USA’s tennis hopes.

Their defeat followed a win in women’s singles by world No. 1 Serena, who joined Madison Keys as the only American women left in the singles draw. The men have also struggled as Steve Johnson is the lone American player left in the men’s draw.

Team USA has fared better in doubles, aside from the Williams’s noticeable loss, as Americans Bethanie Mattek-Sands and CoCo Vandeweghe were victorious in their first-round doubles match. Johnson and Jack Sock also won their match, but Sock suffered his own surprising upset on Saturday in singles. Brian Baker and Rajeev Ram defeated the fourth-seeded French team of Gael Monfils and Jo-Wilfried Tsonga to end the night on a positive note for the Americans.

On Saturday, world No. 6 Venus appeared to be on her way to the second round early in her match against Belgium’s Kirsten Flipkens but faltered to the finish in her three-set defeat.

According to Coach Mary Joe Fernandez, Venus has been struggling with a virus — and she looked out of sorts in both her singles and doubles matches.

The U.S. entered the Olympics with one of its weaker squads since tennis returned as a full medal sport in 1988. The top two men’s singles players, John Isner and Sam Querrey, opted to the skip the Rio Olympics, which does not offer points or prize money, to focus on the U.S. hard-court swing that culminates with the U.S. Open later this month.

Those counting on the Williams sisters as one of  Team USA’s best hopes for Olympic gold in tennis were left trying to make sense of the biggest upset of the Olympics tennis tournament so far.