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39 IMPAIRED SEMEN QUALITY IN TRANS WOMEN 3 MATERIAL AND METHODS Patient selection All trans women seen at the gender identity clinic in the VU University Medical Center (VUmc) between 1972 and 2017 who provided at least one semen sample for cryopreservation before 2018 were identified. People who were under 16 years of age at the time of semen cryopreservation were excluded. This resulted in 260 trans women for the present analyses who, in total, provided 748 semen samples stored in 11 different fertility laboratories in The Netherlands. Study design and clinical data selection The medical charts from included trans women were used to obtain data about their medical history, medication use, prior gender-affirming hormone use, alcohol consumption, smoking, cannabis use, BMI and semen characteristics. For information on BMI, alcohol consumption, smoking and cannabis use in the general Dutch population of similar age, data was obtained from the online database of Statistics Netherlands. 74 The study protocol was assessed by the Ethical Review Board of the VU University Medical Center Amsterdam. It was concluded that the Medical Research Involving Human Subjects Act (WMO) did not apply to this study, and necessity for informed consent was waived because of the retrospective design and the large study population. Laboratory tests Endocrine laboratory data were collected from the hospital registries of VUmc where clinical data, obtained during regular patient care, is stored. The laboratory results included serum concentrations of testosterone, estradiol, LH, and FSH. Counseling and referral for fertility preservation took place before commencing hormonal treatment, thus results from blood obtained by venipuncture at the initiation of hormone treatment were used. Dates of sperm banking were compared with dates of initiation of hormone treatment, accepting an interval of fewer than 120 days for reliable coupling of semen parameters to endocrine data. Semen quality Semen cryopreservation was performed in ISO 15189 accredited fertility laboratories in the Netherlands, all following the Dutch guideline for sperm banks. 75 All trans women were asked to provide semen samples via masturbation after 2 to 5 days of abstinence. The vast majority of trans women provided at least two semen samples for cryopreservation. Semen characteristics were manually assessed at time of semen cryopreservation, prior to the freezing process. Samples were kept at 37 °C before analysis. The semen parameters measured were semen volume, sperm concentration and sperm motility. Volume was determined using a wide-bore volumetric pipette. To assess sperm concentration and motility, a Makler counting chamber (Sefi-Medical Instruments LTD, Haifa, Israel) and phase contrast microscope optics (200-400x) were used. Pre-freeze