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"I dedicate this song to the pope, because I'm a child of God. All of you are also children of God," the 50-year old "Queen of Pop" told the 60,000 fans that flocked to the Italian stop of her "Sticky & Sweet" world tour on Saturday.
Italian newspapers gushed over the singer's electric performance and called the dedication a surprising provocation.
"At the Roman leg of her tour, Madonna didn't miss the opportunity for a provocation that will certainly be discussed," Italy's top newspaper Corriere della Sera said in a front-page report.
Madonna, who comes from a devout Italian Catholic family, has raised the ire of the Catholic Church in the past with sexually charged antics designed to shock.
In 2006, she staged a mock-crucifixion at a concert in Rome to the backdrop of accusations from the Vatican of blasphemy.
The Vatican also condemned her controversial 1989 video for the song "Like a Prayer" that featured burning crosses, statues crying blood and Madonna seducing a black Jesus.
The Vatican later panned a show where she decked the stage out with religious imagery as one of the most "satanic shows in the history of humanity".
Mmm, pwnt?