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Prime Minister Rishi Sunak has said biological sex "really matters" after he was asked about the case of a rapist who was sent to a women's prison.
Isla Bryson was convicted of attacking two women while known as a man called Adam Graham.
One of the victims later said she was sure Bryson was pretending to be trans to "make life easier".
And First Minister Nicola Sturgeon said she was sure Bryson was "almost certainly" faking being trans.
Bryson was moved from Cornton Vale to the male prison estate after a public outcry.
The case was put to the prime minister in an interview with Piers Morgan for Talk TV to mark Mr Sunak's first 100 days in office.
Morgan said the case showed the problem of "limitless gender self-identity".
Mr Sunak said it demonstrated "some of the challenges", but added "we must and should have enormous compassion and tolerance and understanding for those who are questioning their gender and identity".
"But we have to recognise the challenges that that poses, particularly for women's safety," the prime minister said.
"For me… whether it's sex, whether it's women's spaces, whether it's prisons, biological sex really matters."
During the interview, Morgan asked Mr Sunak for his definition of a woman. The prime minister replied "adult human female", but suggested the TV presenter was actually asking about society's handling of people questioning their gender identity.
On Sunday a "pause" was placed on the transfer to women's jails of trans inmates with convictions for violence after it was reported that another transgender woman, Tiffany Scott - who was convicted of stalking a 13-year-old girl before her transition and has a history of violence - was due to be moved to a female prison.
The Bryson case was raised at First Minister's Questions on Thursday by Scottish Conservative leader Douglas Ross on Thursday.
Mr Ross said: "I believe a double rapist, anyone who rapes a woman, is a man. They cannot be considered anything else.
"When a man rapes two women, we don't think that he should be considered a woman just because he says so. We should call out criminals like this who are abusing the system.
"Adam Graham, who wants to be know as Isla Bryson, raped two women. He is an abusive man seeking to exploit loopholes in the government's current policy."
Mr Ross asked the first minister: "Is this double rapist a woman?"
Ms Sturgeon initially said she did not have enough information to say whether Bryson's claim to be a woman was valid or not.
Mr Ross went on to read a quote from one of Bryson's victims, who said: "I don't believe he is truly transgender. I feel as if he has made a mockery out of them using it. As far as I'm concerned, that was to make things easier for himself. I'm sure he is faking it."
The first minister responded: "My feeling is that is almost certainly the case, which is why the key factor in this case is not the individual's claim to be a woman.
"The key and in fact only important factor in this is that the individual is convicted of rape - the individual is a rapist - and that is the factor that should be the deciding one in decisions about how that prisoner is now treated."
Ms Sturgeon went on to say it was "really important" that trans people are not further stigmatised generally.
She added that there were exemptions under the current UK equality law that "even if it wanted to this parliament couldn't change" that enabled trans women to be excluded from some single sex spaces.
Bryson was found guilty last month of raping two women in 2016 and 2019 before she changed gender after being arrested.
While awaiting trail, she enrolled on a beauty course Ayrshire College, where she was known as Annie, and remained there for three months before being asked to leave.
Her classmates were almost exclusively female and much younger than Bryson, and were not aware of the rape allegations.
Potential impact
One former classmate told BBC Scotland last week that she felt "violated" after learning of the crimes Bryson had committed.
Scottish government legislation aimed at allowing people to self-identify their legal sex has been blocked by the UK government over its potential impact on equalities laws.
The UK government said during a Westminster debate on Thursday that it was up to the Scottish government to bring forward a new Gender Recognition Reform bill that addresses the legal issues which caused the bill to be blocked.
Cabinet Office minister Alex Burghart said the decision to block the legislation using what is known as a Section 35 order was used very carefully and reluctantly in order to preserve the balance of powers between Scotland and England.
SNP MP Patrick Grady said the UK government should publish its own amendments to the Bill to make it acceptable and claimed the Conservatives were undermining the Scottish Parliament.