Marilen Benner

CHAPTER 4 94 MATERIALS AND METHODS Experimental model and subject details Peripheral blood was collected from 36 healthy female volunteers (mean age 22.8 years ± 3.4 years). Exclusion criteria were smoking, chronic disease, medication, carrier of an infectious disease and pregnancy. Menstrual blood was provided by 28 healthy women (exclusion criteria: known fertility disorder, potential carrier of an infectious disease, auto-immune disease, drug abuse, or use of hormonal or intra-uterine device contraceptives) with regular menstrual cycle. First and 2 nd trimester material was obtained from discarded placental and decidual tissue upon elective pregnancy termination of healthy women at a local reproductive health clinic. No additional data other than gestational age at time point of termination was acquired. Exclusion criteria were carrier of an infectious disease (active systemic infection), suspected to be a potential carrier of an infectious disease or an increased risk for infection (HIV, Hep B/C, HTLV or similar), auto-immune diseases, drug abuse, intoxication with heavy metals or pesticides. Term placental tissue (>37weeks gestation) was collected from planned cesarean section following uncomplicated pregnancy. Exclusion criteria were gestational age <37 weeks, use of immunosuppressive drugs, biological or antidepressants, HIV positivity, active infection during caesarean section, signs of infection (maternal fever or signs of intrauterine infection), use of antibiotics prior to caesarean section. All gestational samples were assessed visually and excluded for processing in case of signs of infection (discoloration) or excessive blood clots. Written informed consent was obtained for all volunteers donating blood or tissue for this study in accordance with the Dutch Medical Research Involving Human Subject Act (WMO). The study was approved by the local review board (Commissie Mensgebonden Onderzoek region Arnhem-Nijmegen, 42561.091.12, 2017-3253, 2014-232, 2009/004). An overview of all study participants is given in Supplemental Table S1. The amount of individual participants included per experiment is indicated in the according legend as the amount of cells isolated per sample limited the number of experiments carried out simultaneously. METHOD DETAILS Sample collection Peripheral blood was collected using EDTA tubes. For menstrual blood, women were asked to use a menstrual cup (Femmecup Ltd, London, UK) and collect its content every 12h during the first 36h after initiation of menstruation. Contents of the cup were poured in a 30 ml tube containing 10 ml of supplemented RPMI 1640 medium (1 mM pyruvate, 2 mM glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin (all Thermo Fisher Scientific Waltham, USA) 0.3% v/v sodium citrate (Merck Darmstadt, Germany) 10% v/v human pooled serum; HPS, manufactured in-house) for storage at room temperature until processing. After collection, menstrual blood samples were transferred to the lab immediately to undergo processing within max. 24h. First