Marilen Benner

UTERINE LYMPHOCYTES GAIN EXPERIENCE DURING PREGNANCY 37 2 In conclusion, we showed that the immune cell composition of pre-pregnancy endometrium differs from term decidua, but with a similar distribution of Th1, Th2, and Th17 cells. At the end of pregnancy, the uterine immune environment appears to be marked by a tolerogenic phenotype with more experienced T cells and Treg with a potential beneficial phenotype. How exactly the phenotype of the uterine immune cells is shaped and how this differentiation is maintained is unclear, Therefore, in follow-up studies, we aim to explore the influence of different immune triggers on immune cells in the uterine environment before and during pregnancy. This may add insight to our understanding of the pathogenesis of pregnancy complications. MATERIALS AND METHODS Blood and tissue sampling Paired peripheral blood and menstrual blood was collected from 17 healthy women with regular menstrual cycles. Hormones can modulate immune cell responses and change the natural menstrual cycle (28, 29). To avoid any artificial effect on the hormonal balance, none of the menstrual blood donors used any hormonal contraceptives like birth control pill or an intra- uterine device. See Supplementary Table S1 for donor characteristics. 10 ml of peripheral blood was collected in ACD-A tubes. Menstrual blood was collected during the first 36 hours of menstruation using a menstrual cup (Femmecup Ltd, London, UK). Every 12 hours, the sample was decanted from the cup in a 30 ml tube containing 8 ml 10% human pooled serum (HPS) medium [RPMI 1640 medium supplemented with pyruvate (1 mM), glutamax (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml) (Thermo Fisher Scientific, Waltham, USA), 10% HPS (manufactured in-house), and 0.3% sodium citrate (Merck, Darmstadt, Germany)] and stored at room temperature. After 1.5 days, three tubes containing different fractions of menstrual blood were processed immediately to assess lymphocyte composition. Decidua parietalis, the maternal side of the fetal-maternal interface, was obtained from 19 healthy women after uncomplicated term pregnancy. Decidual samples were obtained after delivery by planned elective cesarean section and processed immediately. The study was approved by the institutional review board (Commissie Mensgebonden Onderzoek region Arnhem-Nijmegen, CMO nr. 2009/004) and was performed in accordance with the relevant guidelines and regulations. Samples were obtained from each participant upon written informed consent. Isolation of lymphocytes One ml of peripheral blood was lysed with 25 ml lysis buffer [NH 4 CL + KHCO 3 /Na 4 EDTA (Merck, Darmstadt, Germany) diluted in H 2 O (Versol, Lyon, France)] for 10 min and washed 3x times with PBS (Braun, Melsungen, Germany). These cells were used for surface staining. For intracellular staining, peripheral blood mononuclear cells (PBMC) were isolated by means of density gradient centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway). After isolation,