It throws "Could not verify URL is accessible..."

I have a subdomain ( for which I need a certification. The files for this subdomain are in the Default Web Site of IIS 7.5 and are accessible on a different port than 80. I have stopped the Firewall totally but when I use the Test the first 2 steps are ok but at the end throws:
Could not verify URL is accessible:

Note that on the router I have NAT only on the port concerned and not on the 80. Do I have to do that too?

When you are using http validation (the default) Let’s Encrypt will try check your domain using http (port 80). If you cannot serve a response on port 80 you need to use DNS validation instead (see the Authorization tab), this is a just a limitation in the ACME domain validation process.

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