169 Estradiol and other sex-hormone dynamics in menstrual migraine 8 When a positive result occurred, participants were instructed to contact the coordinating investigator via phone-call or text-message. The day of the LH surge was defined as day 0. Aligning menstrual cycle days to the LH surge allows for the standardization of the luteal phase across all participants, with days 1 to 14 representing the luteal phase and days -1 to -14 representing the follicular phase. Participants were invited to the Leiden University Medical Center at days 1, 2, 3, 8, 10, 12 and 14 after the LH surge for blood collection. After one month of measurements, participants were asked to complete another month. Methods of measurements A standard operation procedure was followed for each measurement. Blood samples were drawn from the antecubital vein while participants rested in a seated position. Blood was allowed to clot and then centrifuged at room temperature for 20 minutes at 622 g (2000 rpm) to separate the plasma. The plasma was aliquoted into 500 µL portions and stored at -80 °C. Upon completion of data collection, samples were transported to Rotterdam Erasmus MC. Biochemical and mass-spectrometry analyses were conducted in an ISO15189 accredited medical laboratory, by lab technologists who were blinded to participant identity, diagnosis, and study day. An in-house developed multiplex MRM ESI-LC-MS/ MS assay was used for high-precision quantification of the serum steroid profile (Waters XEVO TQ-XS, Waters, Wilmington, England). Calibration curves were constructed using standard reference materials (Cerilliant, Round Rock, Texas, USA). Immunoassays were performed using an automated chemiluminescent immunochemistry (Siemens Atellica IM1300, Siemens healthineers, The Hague, NL) according to the manufacturer’s instructions. For all measurements, internal quality Figure 1. Study design
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