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52 C H A P T E R 4 ABSTRACT Background: Transgender women (birth-assigned males, female gender identity) can choose to cryopreserve semen prior to their medical transition, to retain the possibility to parent genetically related offspring later in life. Our previous retrospective study showed that semen quality in transgender women is decreased compared to the general population. The etiology of this impaired semen quality remains largely unknown, but might be related to habitual behavior more typically observed in transgender women, e.g. the desire to hide their testicles due to genital dysphoria. Therefore, we decided to conduct a consecutive study with prospectively obtained data on behavior and lifestyle in transgender women. Objective: To study the influence of a lowejaculation frequency,wearing tight undergarment, and bringing the testes in the inguinal position (tucking), on semen quality in transgender women at time of fertility preservation. Study design: In this cohort study, transgender women were included between May 2018 and September 2020, at time of fertility counseling, prior to the start of hormonal treatment. Data were collected on demographics, lifestyle factors, medical history, endocrine laboratory results, and semen parameters. Semen parameters were categorized using reference values for human semen of the World Health Organization and compared with semen quality in the general population. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using multivariable logistic regression analysis to assess the impact of tucking, wearing tight undergarment, and a low ejaculation frequency on semen quality, correcting for potential confounders. Results: Overall, 113 transgender women were included. Median semen parameters were significantly decreased compared to the general population. Crude logistic regression analyses showed an association between always wearing tight undergarment (OR 3.06, 95% CI 1.11-8.49), and extensive tucking (OR 6.09, 95% CI1.54-24.01), on having a total motile sperm count below 5 million. Multivariable analyses showed that the association with tucking was independent of demographic factors, lifestyle factors and medical history (OR 7.95, 95% CI 1.66-37.99). However, this was not the case for the association with always wearing tight undergarment (OR 2.89, 95% CI 0.95-8.82). Ejaculation frequency did not influence total motile sperm count. Conclusions: Behavioral factors, including wearing tight undergarment and extensive tucking, may contribute to the lower semen quality in transgender women. These results enable optimization of fertility counseling on how to adjust lifestyle before pursuing semen cryopreservation.