Wisconsin, Kentucky, Nebraska and Kansas joined Texas in a new lawsuit, filed with the Christian Medical and Dental Association, and Franciscan Alliance of Indiana.
The states object to federal guidance on transgender students' rights, including the right to use facilities matching their gender identity.
The businessman inches closer to clinching the nomination.
Hillary Clinton still holds a big lead in the delegate race for the Democratic nomination.
Transgender students in Nebraska must now obtain approval from their school and a separate committee of doctors and psychiatrists to participate in activities that correspond with their true gender.
Failing to list both parents is clearly unconstitutional, so a federal court should order the state to do so without a trial, the couples say.
The proposal would establish a committee to decide who's trans and assure that birth gender generally supersedes gender identity.