Distribution of decidual natural killer cells and macrophages in the neighbourhood of the trophoblast invasion front: a quantitative evaluation

Do decidual natural killer (dNK) cells and decidual macrophages (dMph) become enriched in the vicinity of the trophoblast invasion front?Morphometric image analysis and areal cell density calculations, which excluded observer bias, showed an enrichment of decidual leukocytes in the neighbourhood of the trophoblast invasion front.In previous studies, the number of decidual leukocytes was visually counted in medium- or high power fields. These methods, however, cannot reveal the exact spatial relationship between leukocytes and invasive trophoblast cells, and are therefore prone to subjective errors. Thus, a more objective approach is required.Applying a new method of morphometric image analysis, leukocyte populations were studied in human tissue fragments derived from first trimester placentation sites (n = 7) as well as in co-cultures of first trimester decidual tissue with placental villi of the same pregnancy representing an appropriate in vitro model of trophoblast invasion (n = 15).First trimester decidual tissue was obtained from women undergoing elective terminations of pregnancy at 7-10 weeks of gestational age. Tissue sections were double-stained immunohistochemically for markers of dNK cells or dMph on one hand, and for invasive extravillous trophoblast cells on the other. To analyse the distribution of leukocytes, distinct cell compartments as well as cell neighbourhood areas were defined. Finally, relative areal cell densities were calculated and these data were compared with those of an in vitro model of trophoblast invasion as well as with tissue fragments derived from decidua parietalis without trophoblast cells.At first trimester placentation sites, a higher density of dNK cells as well as of dMph was found in close proximity to the invasive trophoblast (P ≤ 0.01), compared with the average areal cell density of decidual leukocytes in the tissue with exclusion of the trophoblast. The highest areal cell density of leukocytes was determined up to a distance of 20 μm from the trophoblast cells, whereas in more distant regions it was even lower than average, indicating a migration of these leukocytes towards the trophoblast invasion front. In the three-dimensional co-culture model, however, we found an enrichment of dMph (P ≤ 0.01) but not of dNK cells (P > 0,05) in the neighbourhood of the invasive trophoblast.The morphometric image analysis depends on intense immunohistochemical staining that is free of background and cross-reactivity.The presented method will be useful not only for the investigation of recurrent miscarriage but also in the fields of tumour immunology and inflammation.The study was supported by the European Commission (Network of Excellence 'The Control of Embryo Implantation (EMBIC)', FP6-512040, lead researcher: P.S.), and by the Franz Lanyar Foundation of the Medical University of Graz, Austria (Grant #347). None of the authors declared a conflict of interests.