A blinded multicenter investigation: Accentuated NK lymphocyte CD335 (NKp46) expression predicts reproductive failures after IVF.

The peripheral blood NK cell diversity is highly complex. Recent studies have described more than a thousand phenotypes sharing NK cell receptors (NKRs), across the leukocyte lineages. Previously, we have found that accentuated NK p46 phenotype has prognostic value for NK cytotoxicity status, and is characteristic for patients with recurrent implantation failure (RIF). In a blinded investigation we studied blood samples from IVF women before embryo transfer (pre-implantation genetic tested [PGT] embryos n = 116; not tested embryos n = 219). We studied NKp46 expression by flow cytometry and anti-cardiolipin antibody (aCL) levels. aCL results were transmitted to the clinic but NKp46 expression was blinded (for us and for the clinic) and not analyzed before termination of the study (end of last pregnancy). Association of NKp46 phenotype with clinical pregnancy rate (CPR), pregnancy failure (PF) rate and life birth rate (LBR) were analyzed. aCL positive and IvIg treated cases were excluded. IVF success was dependent on p46 NK phenotype in patients with PGT embryos. Elevated p46 expression on NK (>93%) as well as decreased (<66%) significantly reduce CPR (OR 12.7 and 3.8) without affecting pregnancy failure frequency. Both accentuations (taken together) resulted in a significant reduction of LBR (OR 3.9 p = 0.019) compared with non-accentuated phenotypes (p46 levels 66-93%). Elevated NK cell levels (>14.5% weakly) were associated with PF (OR 3.1 p = 0.069), but not significantly with reduced LBR. In contrast, numbers of NKCD335+ lymphocytes (>11.5%) were a significant predictor of PF (OR-4.0 p<0.05) and decreased LBR (OR 2.1 p = 0.06). At the same time, accentuated numbers of NKCD335neg lymphocytes (<0.7 and >4%) were also associated with decreased LBR (OR 2,65 p = 0.05). In patients with NKCD335++ numbers (<5 and >21%), we found a weakly association with IVF failure. We found similar associations in IVF patients without PGT -A but at lower significance levels regardless the higher number of patients. Impact of NKp46 phenotype for IVF success was significant in patients with donor's ET and almost imperceptible in patients > 35y.o. with own embryo transfer. Accentuated increased or decreased CD335 expression on NK was associated with embryo implantation failure. Balanced CD335 levels form a condition favorable for implantation. Elevated numbers of p46+NK (CD3-CD56+CD335+) predicts pregnancy failures at higher significance levels than elevated NK cell numbers. Elevated numbers of p46negNK (CD3-CD56+CD335-) indicate reduced LBR. Accentuation of p46 expression on NK cells is associated with reproductive failures. In combination with PGD it provides a powerful prediction algorithm and treatment option.