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.

Expression of natural cytotoxicity receptor NKp46 on peripheral blood natural killer cells in women with a history of recurrent implantation failures.

AIM: The peripheral blood natural killer (NK) cells diversity is highly complex; recent studies described more than a thousand phenotypes sharing NK cell receptors, across the leukocyte lineages. In this study, we investigated the expression of NKp46 in peripheral blood NK cells in women with a history of recurrent implantation failures (RIF) with euploid embryos with pre-implantation genetic diagnosis (PGD) and control group (donors of oocytes and surrogate mothers). METHODS: The expression of NKp46 in peripheral blood lymphocytes and NK cells from women with RIF (n = 57) and control group (n = 50) was analyzed with three-color flow cytometry. RESULTS: The percentage of NKp46+ NK cells was significantly higher in women with RIF compare with the control group and high amount of NKp46+ NK cells (>13% of total lymphocytes) was a poor prognostic factor for embryo implantation. Also, women with RIF had a low amount of NKp46neg NK cells, which was a negative prognostic factor for embryo implantation. The analysis of NK subpopulations, on the basis of NKp46 expression, also revealed that NKp46neg NK in low amounts (<20% of NK cells) and NKp46dim in high amounts (>50% of NK cells) are also negative prognostic factors for embryo implantation. CONCLUSION: Our results support the clinical significance of the NKp46 expression on NK cells in women with RIF. We suggest that the low level of NKp46neg subset in women with RIF may be a result of an imbalance in the differential development of ILC subsets toward cytotoxic ILC (NK cells), which in turn is a negative condition for successful embryo implantation.

Natural killer frequency determines natural killer cytotoxicity directly in accentuated zones and indirectly in "moderate-to-normal frequency" segment.

Natural killer (NK) frequency and NK cytotoxicity (NKc) are key determining factors of a clinical outcome. In our previous study, we showed the prognostic clinical significance of immune parameters when they are beyond the optimal range (accentuated). In this study, we attempted to explain the disparity of accentuated but physiologically and immunologically normal NK parameters that might serve as negative clinical prognostics indications of failed pregnancies. We have analyzed NK%, NKc levels, and their reciprocal correlation in 2,804 patients with reproductive failures. In the entire clinical population, NK% correlates with NKc. Interestingly, we found this relationship to be strongly dependent on NK level's status. NK%-NKc correlation was the strongest (r = 0.2021, p < 0.0001) in a patient group with high NK% (> 17.5%). Patients with NK% between 15-17.5% manifested lower but still significant correlation NK%-NKc (r = 0.1213, p = 0.0155). Additionally, significant correlation (r = 0.2689, p < < 0.0001) between NK% and NKc was observed in a group of patients with NK levels < 7% (1.7-7%). While patients' groups with NK% (7-15%) did not reveal NK%-NKc association. This led us to hypothesize that the qualitative-quantitative status of NK population is responsible for their cytotoxic activity. Consistent with our hypothesis, the "balanced zone" NK% is tightly controlled, and thus does not correlate directly with NKc. In contrast, the "accentuated zones" of NK% escape this control and directly affecting NKc. Demonstrated phenomena supports our idea about the clinical significance of immune accentuation and explains its novel physiological role.

Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro.

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses. The monitoring of NK cells function is important in various physiological and pathological conditions. Different approaches have been used to directly or indirectly evaluate NK cells activities. The purpose of this study was to investigate the correlation between the number of NK cells and cytotoxic activity of NK cells and to determine whether NKp46+NK cells reflect NK cytotoxicity status. In our study, we retrospectively analyzed laboratory data on NK cytotoxicity and NK lymphocyte levels of 4896 infertile women which underwent routine immunology investigation after IVF failures. In healthy women, NKp46 expression was assessed on NK cells (n = 214) and cytotoxicity activity was evaluated with regard to NKp46 expression. We found that despite a significant correlation coefficient (n = 4689, r = 0.447), the correlation with cytotoxicity is maintained only within the zones with a low or high NK cells frequency. NK cells frequency has no significant prognostic value for their cytotoxicity - within the medium NK frequency zone the samples may have any cytotoxicity, both reduced and elevated. However, our data demonstrate that NKp46+NK cells frequency correlates with cytotoxicity activity even more significantly than the NK cells frequency (n = 214, r = 0.67 and r = 0.62, respectively) and has significant prognostic value for the abnormal NK cytotoxicity status indications, both low and increased. Our results further support an important role of NKp46 in NK cells killing and afford grounds for using the measurement of the NKp46+NK cells frequency as an alternative method for abnormal NK cytotoxicity status indication, which is responsive, simple and reliable.

Repeated cupping manipulation temporary decreases natural killer lymphocyte frequency, activity and cytotoxicity.

OBJECTIVE: Elevated natural killer lymphocyte cytotoxicity (NKc) has been linked with reproductive problems in women. Here we evaluate the potential benefit of cupping therapy (CT) in reproduction-related immune responses. METHODS: This was a pilot clinical study. Participants were healthy female volunteers (n = 23) with elevated NKc, and received repeated CT 3 times over 5 d (inner pressure 40-50 kPa, 40 min; 12-15 cups). Lymphocyte subsets, NKc and NK lymphocyte activity (NKa) were measured in blood on day 0 (initial levels, before the first treatment) and days 3, 10 and 17 after the last CT treatment, using the K562-stimulated CD69 expression assay. RESULTS: As a result of CT manipulations NKa was reduced on days 3 and 10, and NK percentage was reduced on day 10. NKc was most sensitive to CT treatment, resulting in their decreased counts at 3, 10 and 17 d post CT. CT treatment decreased NKc in the majority of individuals (87%), but the magnitude of the effect was variable. Out of 23 subjects 9 (39.1%) had a 2-3 fold decrease of NKc on days 3, 10 and 17; 11 (47.8%) started to show a decrease in NKc later, or more quickly returned to base levels; and only 3 (13%) subjects displayed no effect of CT on NKc. Expectedly, no changes in T-cell subsets (CD3CD4, CD3CD8, HLADR, CD158a) were observed after CT. CONCLUSION: CT decreased NK cell numbers, their activity and cytotoxicity. Low cost, safety, non-invasive nature and ease of administration make CT a promising approach for NKc down-regulation.

Peripheral blood natural killer cells activation status determined by CD69 upregulation predicts implantation outcome in IVF.

PROBLEM: NK lymphocytes play critical yet poorly defined role in implantation and during development in early pregnancy. METHODS OF STUDY: Recently, we showed that the proportion of NK that expressed CD69⁺ after incubation with K562 (CD69(stim)) cells reflected the NK population excitation potential. In the present study, we investigate the significance of NK activation levels in predicting embryo implantation. RESULTS: A qualitative analysis of values distribution in two groups showed that 25/33 (75.8%) women who became pregnant had CD69(stim) levels that were >30 but <60% (conditionally normal zone). In contrast, CD69(stim) levels in patients who failed to become pregnant were either elevated, as in 10/51 (19.6%), or reduced, as in 20/51 (39.2%) of the patients. Accentuated CD69(stim) levels were predictive for implantation failure, extremely significant for decreased (OR 6.9, p=0.0004) and not quite significant for increased CD69(stim) levels (OR 3.9, p=0.062). Accordingly, conditionally normal CD69(stim) levels were favourable for implantation (OR 4.46, p=0.0032). CONCLUSION: We confirm that actual peripheral blood natural killer cells activation status have an influence on embryo implantation. We showed that exactly normal NK cell activity predicting successful implantation.