Evaluation of peripheral NK tests offered to women with recurrent pregnancy loss and a search for novel candidate biomarkers

Recurrent pregnancy loss (RPL) is a common condition of largely undetermined pathogenesis. There is prior evidence of association with immune dysregulation linked to elevated NK cell levels and cytotoxicity. However, experimental findings remain contentious, hindering clinical adoption of immunological testing. Given their importance in healthy pregnancy, this study set out to determine the clinical utility of NK cell assays in 100 non-pregnant women with RPL and 80 healthy control women and establish an exploratory mass cytometry panel for in-depth NK phenotyping. As previously described, peripheral NK cell elevation was observed with RPL. The augmented NK cell cytotoxicity, often referenced, was undetectable, although enhanced degranulation was observed. Reduced cytolytic molecule secretion by PBMCs was seen in RPL, possibly counterbalancing the increased NK cell degranulation. Mass cytometry was employed for the detailed investigation of NK cell phenotype, focused on inhibitory and activating receptor expression. Augmented prevalence of CD57⁺ mature cytotoxic NK cells was present in the RPL cohort. This was accompanied by elevated prevalence of subsets lacking inhibitory receptor expression, indicating enhanced NK cell responsiveness to activating signalling. Additionally, CXCR3/CXCR4⁺ subset reduction, suggested potential uterine migration defects. This extensive analysis of peripheral NK cells in RPL has revealed significant dysregulation affecting both total number and potential activity. The extent to which this dysregulation is reflected in utero requires further examination. Current findings will be used to guide subsequent investigations on paired peripheral and endometrial samples as well as biomarker discovery to improve our capacity to estimate risk of a following loss.