Marilen Benner

IMMUNE PROFILES IDENTIFY RECURRENT PREGNANCY LOSS 117 5 allows analysis of large cohorts. More women suffering from RPL have to be enrolled to account for the heterogeneity of this cohort. Combined with inclusion of a validation cohort, this will ensure generality of results to enable translation towards diagnostic tests. This is of special interest for RPL, a condition that not only needs proper diagnosis, but would benefit from repeated sampling in order to assess the success of intervention strategies. To both ends, classification needs to be robust and accurate, and deserves further assessment. Taken together, RPL patients present with a dysregulated immune environment, systemically and within the uterus. Cohort classification of possible diagnostic value cannot rely on individual immune cell frequencies but rather depends on a combination of immune cell subsets. The non- invasive source of menstrual blood, allowing to investigate important regulatory mechanisms, holds many opportunities for the assessment and monitoring of reproductive health. MATERIALS AND METHODS Sample collection Peripheral and menstrual blood was collected from women without known disorders of reproduction, with regular menstrual cycles, and women suffering from recurrent pregnancy loss (RPL). Characteristics of women included in this study are documented in Table 1. Exclusion criteria were autoimmune diseases, smoking and current use of an intra-uterine device or hormonal contraceptives. 10 ml of PB was collected in ACD-A tubes. Women used a menstrual cup (Femmecup Ltd, London, UK) for 3 12h intervals during the first 36h of menstruation. Menstrual effluent was collected in supplemented RPMI 1640 media (pyruvate (1 mM), glutamax (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) (all Thermo Fisher Scientific, Waltham, USA), 10% human pooled serum (HPS, manufactured in-house), and 0.3% sodium citrate (Merck, Darmstadt, Germany) for storage at room temperate until processing. Samples were transferred to the lab for immediate processing, within max. 24h after the last collection. Written informed consent was obtained for all volunteers in accordance with the Dutch Medical Research Involving Human Subject Act (Commissie Mensgebonden Onderzoek Arnhem-Nijmegen, Nr 2017-3256). Leucocyte isolation Leucocytes from PB were isolated through density gradient centrifugation (Lymphoprep, Axis- Shield PoC AS, Oslo, Norway). MB was processed as described previously (25, 37). Briefly, menstrual blood derived cells were obtained after granulocyte depletion using RosetteSep (Stemcell technologies Inc, Vancouver, Canada) followed by density gradient centrifugation (Lymphoprep).