Changes in the immunophenotype of endometrium during implantation window receptivity formation in healthy fertile women.

INTRODUCTION: Endometrial receptivity is largely determined by the immunophenotype of endometrium, especially uterine NK-cells (uNK). Immune component is directly involved in the formation of favourable microenvironment for the blastocyst implantation and placenta formation, but the way it changes during the maturation of endometrial tissue in healthy fertile women is still underexplored. METHODS: The endometrium was collected from 47 healthy oocyte donors after controlled ovarian stimulation: 23 women on the day of oocytes retrieval (OR) and 24 women on the term of implantation window (IW). The OR group was analysed, published previously and used as a comparison group to show the dynamic of changes. Isolated endometrial lymphocytes and peripheral blood samples were stained with monoclonal antibodies and analysed according to the three-color flow cytometry protocol. RESULTS: The proportion of NK-cells (CD3-CD56+) in endometrium grew significantly in the implantation window compared to the oocytes retrieval day. NK-cells acquired a more differentiated phenotype from the day of OR until IW: the expression of CD8 and CD158a significantly increased, while the expression of HLA-DR significantly decreased. Significant correlations between peripheral blood and endometrial NK-cells were found in CD8 expression during OR and IW, CD335(p46)neg and CD335(p46)++ subsets during IW term. DISCUSSION: Immunophenotype of receptive endometrium forms due to the accumulation of uNK-cells, which actively proliferate, become mature, differentiative, and ready to meet the embryo. Endometrial immunophenotype is peculiar and specific but not autonomic and isolated. Differentiation (CD8 on NK-cells), and activity (p46 on NK-cells) of peripheral blood lymphocytes is reflected in endometrial lymphocytes profile, and therefore the research of peripheral blood immunophenotype is relevant.

A Blinded Investigation: Accentuated NK Lymphocyte CD335 (NKp46) Expression Predicts Pregnancy Failures.

AIM: NKp46 is an NK cell receptor uniquely expressed by NK cells and a small subset of innate lymphoid cells. In our previous studies, we suggested a tight connection between the activity of NK cells and the expression of NKp46 and supported the clinical significance of NKp46 expression in NK cells in women with reproductive failures. In this study, we investigated the expression of NKp46 in NK cells in the peripheral blood of women in early pregnancy and analyzed its association with pregnancy loss. METHODS: In a blinded study, we examined blood samples and analyzed the subsequent pregnancy outcomes from 98 early pregnant women (5th-7th week of gestation-w.g.) and 66 women in the 11th-13th week of pregnancy who served as controls. We studied the expression of NKp46 and the levels of anti-cardiolipin antibodies (aCL). The results of aCL were shared with the clinic, while the expression of NKp46 was blinded and not analyzed until the end of the study. RESULTS: A misbalance in the NKp46+NK cells subpopulations was associated with an unfavorable ongoing pregnancy. A decreased level of NKp46high cells (<14%) was strongly associated with miscarriage. A decreased level of the double-bright subpopulation (NKp46hightCD56++) also was a negative prognostic factor for the pregnancy course, but its increased level (>4%) was strongly associated with a successful pregnancy course. CONCLUSIONS: Our results showed that accentuated levels of NKp46+NK cells lead to a negative prognosis for early pregnancy courses in women.

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.