Peripheral cytokines as candidate biomarkers for recurrent pregnancy loss.

Graphical Abstract: Abstract: A current working hypothesis in recurrent pregnancy loss (RPL) suggests immune dysregulation, underlain by a pro-inflammatory cytokine shift in TH cells. NK cells, which are primary immune effectors throughout pregnancy, also orchestrate immune activity via cytokine secretion, which appears aberrant in women with RPL. Experimental findings remain contentious, hindering clinical adoption of cytokine testing, despite increasing pressure from clinicians and couples affected. This study aims to identify peripheral cytokine biomarkers that could be utilized for the stratification of women with RPL displaying underlying immune dysregulation. Peripheral blood samples from non-pregnant women with RPL (n = 99) and controls (n = 80) were assessed for TH and NK cell cytokine production via flow cytometry. Cytokine secretion by peripheral blood mononuclear cells was examined via ELISA. Elevated pro-/anti-inflammatory cytokine-producing TH cell ratios were detected in RPL (P < 0.0001). This was, however, accompanied by reduced pro-/anti-inflammatory cytokine expression within individual cells (IL-2/IL-10: P < 0.0001, IFN-γ/IL-10: P < 0.0001, TNF-α/IL-10: P = 0.0136, IL-17A/IL-10: P < 0.0001). NK cell cytokine production appeared deficient overall in women with RPL (TNF-α+ NKs: P < 0.0001, IL-10+ NKs: P = 0.0006). Interestingly, a pro-inflammatory shift was evident when cytokine secretion was investigated (TNF-α/IL-5: P = 0.0120, IL-2/IL-13: P = 0.0105, IFN-γ/IL-13: P = 0.0040, TNF-α/IL-13: P < 0.0001). A pro-inflammatory shift in cytokine secretion was identified in women with RPL, indicating systemic cytokine dysregulation. Peripheral cytokine levels emerged as discriminatory and, thus, upon validation, may present valuable biomarkers in the identification of immune-associated RPL. Further research on the functional effects of cytokine imbalance in utero is required to strengthen the pathogenic relevance of these findings. Lay summary: Pregnancy losses (miscarriages) that happen repeatedly may be linked to an overactive immune system. We studied proteins that are used as signals to/from immune cells, called 'cytokines', in blood from women with repeated miscarriages and women without this history. We measured the production of these molecules by two immune cell types that are key in pregnancy: T helper cells and natural killer cells and their total levels in blood. In women with miscarriages, more T helper cells produced immune-activating over immune-suppressing cytokines. Natural killer cells made fewer cytokines overall. When we looked at their total levels in the blood, more immune-activating cytokines were seen. Our findings point to a change towards more immune activation in women with multiple miscarriages. After examining this further, cytokines could be used as a test to identify women who are more likely to benefit from treatments that help balance the immune system.