I think the rule of thumb is if you see a bathroom use it! There was a large bathroom right inside the Vatican entrance and I don't remember if there were others, As someone mentioned just let your tour guide know, or maybe email them before the tour to make sure there are restrooms throughout the site you are visiting to see if this tour is the right fit.
I was once on an all-day bus tour and they specifically said there are no restroom on the bus, to please use the restrooms at the sites we visited. On the 3 hour straight shot back to town, there was one gentleman who had to have the bus stop 3 times due to having to use the potty. The first time the group was sympathetic and understanding, but the 2nd and 3rd time, not so much. I don't know if he should have packed some immodium or what, but the bus driver and tour operator had to figure out where some bathrooms were and some of them were really shady looking areas, like maybe rest areas. It delayed our return by about 45 min. He did not apologize to the group nor did he leave a tip for the driver or guide. Adventures in Travel!!!