Main findings In chapter 2 we showed that estrone concentrations are not associated with change in body fat or breast development or in trans women receiving either oral or transdermal estradiol. Moreover, no difference in clinical outcomes was observed between oral and transdermal administration of estradiol, despite the fact that oral estradiol was clearly associated with much higher estrone levels compared with transdermal estradiol. This suggests that only estradiol and not estrone exerts effect on body fat distribution. In chapter 3 we demonstrated that in trans women, suppressing testosterone and increasing estradiol results in an increase in subcutaneous fat and a decrease in VAT/SAT ratio and liver fat. In trans men, we showed that the increase in testosterone concentrations in combination with lower estradiol concentrations results in an increase in visceral fat, VAT/ SAT ratio and liver fat. In chapter 4 we demonstrated that DXA is a valid method to estimate visceral fat in trans women and trans men at baseline and after 12 months of hormone treatment. However, the change in visceral fat after 12 months of hormone treatment is underestimated by DXA, compared to MRI. In chapter 5, we demonstrated a rapid increase in bone marrow fat fraction after testosterone and estradiol suppression in trans women, followed by a rapid decrease after start of estradiol treatment. In trans men, on the other hand, we observed a rapid increase in bone marrow fat fraction after testosterone treatment and suppression of estradiol by triptorelin and anastrozole. In trans men without anastrozole, the bone marrow fat fraction increased only after 12 weeks and decreased to baseline after 52 weeks. Together these results suggest that estradiol and not testosterone is the major sex steroid regulating the bone marrow fat fraction in both women and men. In chapter 6 we demonstrated that this effect of estradiol on bone marrow fat is not caused by a change in number of adipocytes, lipid droplet area or adipokine expression. The effect of estradiol on bone marrow fat might be exerted earlier in the differentiation of adipocytes or via other cell types. Finally, in chapter 7 we observed that gender affirming hormone treatment induces an increase in cheek tissue in trans women and a decrease in cheek tissue in trans men. The cheeks consist mostly of subcutaneous adipose tissue and therefore we can conclude that sex steroids exert the same effect on subcutaneous tissue in the abdomen and the face. Conclusions In conclusion, this thesis has shown that in trans women, suppressing testosterone and increasing estradiol results in an increase in subcutaneous fat, both abdominal and facial; and a decrease in VAT/SAT ratio, liver fat and bone marrow fat. In trans men, an increase in testosterone concentrations in combination with a decrease in estradiol concentrations results in an increase in visceral fat, VAT/SAT ratio, liver fat and bone marrow fat, and no significant changes in subcutaneous fat. These results suggest that estradiol is the major sex steroid regulating body fat distribution in women and men. However, estradiol does not seem to directly affect mature adipocytes with respect to adipocyte number, lipid droplet area or adipokine expression. The effect of testosterone on body fat distribution acts mainly through the aromatization of testosterone into estradiol.