Iris de Nie

54 C H A P T E R 4 MATERIALS AND METHODS Study design and patient selection A cohort studywas conducted at the Center of Expertise on Gender Dysphoria of Amsterdam UMC. Transgender women above the age of 16 years, who were referred to a fertility specialist and decided to pursue fertility preservation between May 2018 and September 2020, were asked for participation. The Ethical Review Board of the VU University Medical Center Amsterdam concluded that the Medical Research Involving Human Subjects Act (WMO) did not apply to this study. The study protocol was registered in the Netherlands Trial Register with registration number NTR7185. Data collection During the first visit with a fertilityspecialist, informationwas collected on carefullyselected factors with a potential negative impact on semen quality, based on literature assessing the influence of lifestyle on semen quality in cisgender men, and behavioral factors more typically observed in transgender women. Data included demographics, lifestyle factors, medical history, tucking, wearing of tight undergarment, and ejaculation frequency. Blood samples were taken for serum testosterone and FSH concentrations. Subsequently, participants were referred to a regional fertility clinic for semen cryopreservation. Semen parameters were collected after completion of the semen cryopreservation process. Laboratory tests Semen cryopreservation was performed in ISO 15189 accredited fertility laboratories, following the Dutch guideline for sperm banks. 75 Transgender women were advised to collect a sperm sample after 2-5 days of ejaculatory abstinence. Prior to the freezing process, semen characteristics (semen volume, sperm concentration and sperm motility) were manually assessed by qualified laboratory technicians. Volume was determined using a wide-bore volumetric pipette, and sperm concentration and motility were assessed using a Makler counting chamber (Sefi-Medical Instruments LTD, Haifa, Israel) and phase contrast microscope optics (200–400x). Pre-freeze and post-thaw sperm motility were classified using a three-category scheme: progressive motile, motile and immotile. Total motile sperm count (TMSC) was calculated by multiplying the sample volume (ml), sperm concentration per ml and the progressive motility. To prepare the samples for cryopreservation, ejaculates were diluted 1:1 and mixed thoroughly for at least 10 min with medium containing glycerol or egg yolk (TYB, Irvine Scientific) as cryoprotectant and put in 0.3 or 0.5 ml vials. Vials were put upright just above liquid nitrogen for gradual crystallization in nitrogen vapor. After total crystallization, straws were stored in vapor phase nitrogen tanks. Half of the fertility laboratories also assessed post-thaw quality by thawing a vial after 24–72 hours and assessing the semen characteristics again. Some of these laboratories assessed post-thaw quality of all semen samples, others assessed only one of the provided semen samples. The predicted most appropriate reproductive technique was determined by the post-thaw TMSC per vial. In