That moment is the definitive point where the player realizes that, even after surviving, Akane's life will never be the same again. It's bad enough that she was traumatized by being kidnapped, but the second half of her situation is because of an administrative oversight that put her in the wrong group!
Aoi's face when he finds his sister's body in the incinerator in the true ending, though Akane's death is subsequently undone, and Seven's story wherein Aoi found his sister's body may have been...less than honest.
Young Junpei and Akane's farewell on the hilltop in the same ending.
The Safe Ending: Snake, upon learning of Clover's murder at Ace's hands, endures getting shot six times in order to restrain Ace long enough for them both to die in the incinerator. Part of it is to atone for not being able to protect her, and partially so Junpei, Seven, and Lotus can get away, despite the fact that all three of them are begging him not to.
Consider the way everyone reacts to Santa taking June hostage. Clover is especially hurt, since Santa knew about the leaf words that gave her hope. Santa's response is to threaten to put a bullet in her head, spit flying from his mouth. Of course, it's all part of the plan, but that doesn't make it any easier.
Santa's conversations with Junpei become heartbreaking in hindsight, particularly considering that Akane is merged with Junpei's consciousness. His Good Santa/Bad Santa story takes on whole new meaning after the ending. He seems to be asking Akane if what they're doing is right. Even more normal conversations, such as the one about how he played the stock market, is harder knowing that he's telling Akane that everything went to plan along with trying to give Junpei clues.
The Safe Ending has a retroactive one that players won't understand the first time around.
Zero: No. You misunderstand. You haven't lost. I havelost.
Should you choose to force everyone to go through Door 3 regardless of this leaving more people behind than was intended, the result will be a short but intense life-or-death race to the door between Santa, and Lotus and Clover. The former wins and as they go through the door, Junpei takes one last look behind him, the defeat and desperation clear on Lotus and Clover's faces. Particularly harsh in case of Clover, who already has a Heroic B.S.O.D. as her brother is missing and most likely dead, and now you have seemingly doomed her, too.
Ace's motives. He has never truly been a bad person or a mad scientist - just the tired man willing to live a full social life.