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.

HCMV seropositivity is associated with specific proinflammatory immune phenotype in women with implantation failure.

HCMV coevolved with humans for millions of years and is now one of the most widespread infections worldwide. For a long time HCMV seropositivity was considered a safe clinical condition. In recent decades both clinical observations and research results indicated that the very presence of HCMV in human organism specifically influences immune system and may affect reproduction as a process greatly dependent on immune cells function. Anti-HCMV IgG, IgG avidity, lymphocyte subsets as well as NK cytotoxicity was investigated in 470 infertile women who were eligible for IVF/ET. 419 patients were IgG anti-HCMV-positive (HCMV-seropositive) and only 51 (10.8 %) were IgG anti- HCMV-negative (HCMV-seronegative). There was not a single case of clinically significant level of low-avidity IgG. HCMV-seropositive patients had significantly increased levels of HLA-DR expression on T-lymphocytes (both on CD3CD8 and especially on CD3CD4 subsets) and HLA-DR expression on NK-lymphocytes (CD56+CD3-), increased levels of NKT-like cells (CD3+CD8+CD56+) but decreased levels of CD8 + NK lymphocytes compared to HCMV-seronegative patients. That difference was caused by significant numbers of individuals with deviated "accentuated" immune phenotypes in HCMV seropositive patients. The latter had increased (>7.5 %) levels of HLA-DR expression on T helpers in 136 cases from 419 (32.4 %) while in HCMV-seronegative group this accentuation was observed only in 3 of 51 patients (5.8 %), (OR -5.9, p < 0.0003). The number of cases with significantly increased CD56 expression on Tc lymphocytes, HLA-DR on NK and decrease of CD8-positive NK cells was more often observed in HCMV-seropositive group compared to seronegative. Thus, possibly HCMV seropositivity specifically influences immune system and results in pro-inflammatory phenotype formation in part of infected population. It was found that accentuations in immune phenotype of HCMV-seropositive women are very similar to previously described in association with reproductive failures but without HCMV serostatus taken into account.

Comparison of T and NK lymphocyte subsets between human endometrial tissue and peripheral blood.

Immune profiles in endometrium may be changed in patients with IVF failure and its possible correlations with immune parameters in peripheral blood are important for the diagnostic approach. Such correlations in healthy women are unknown and have been studied in the present research. The expression of CD56, CD158a, HLA DR, CD69 in T lymphocytes, CD4 T lymphocytes, CD8+ T lymphocytes and NK cells were studied by flow-cytometry in endometrium and peripheral blood in healthy 24 donors of oocytes aged 25-32 years. Levels of T lymphocyte and T helper cells were lower in endometrium and no differences in CD8 T lymphocytes were registered between endometrium and peripheral blood. The expression of HLA DR and especially CD69 was higher in CD3, CD4, CD8 T cells in endometrium in comparison with peripheral blood. The endometrium lymphocyte population was enriched by NK cells that were generally CD56++ with a higher expression of HLA DR and almost in total were CD69 positive. Strong positive correlations of CD8 expression in NK cells (r = 0.6478, p < 0.001) and HLA DR expression in CD8 T cells (r = 0.6107, p < 0.01) between peripheral blood and endometrium were registered in fertile women. The endometrial CD56 expression in CD8+ T cells negatively correlated with endometrial CD8 expression in NK cells (r = -0.5252, p < 0.01) which possibly reflected a suppressive and regulating mechanism in the endometrium. CD8+ NK cells and HLA DR+ CD8 T cells in endometrium were related to the same subsets in peripheral blood.

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.

Multiple immune deviations predictive for IVF failure as possible markers for IVIG therapy.

Recently we have shown that immune deviations (ID) may predict IVF failure. Benefit from IVIG therapy was observed in 115 women with repeated IVF failure according to proposed multiple ID that appeared unfavorable for implantation and live birth. Group of 123 women with repeated IVF failure without IVIG therapy was compared with former group. Immune phenotype and NK activity of peripheral blood lymphocytes were studied by flow cytometry. Potentially predictive for IVF failure ID included elevated expression of CD56, CD158a in T lymphocytes, decreased levels of CD4T lymphocytes, up-regulated expression of HLA DR in CD8+ T cells and NK cells, elevated number of NK cells and increased NK cytotoxicity, increased or decreased expression of CD158a and CD8 in NK cells. Three or more ID may predict implantation failure to a greater degree than one or two ID. In women receiving IVIG in subgroups with 0-1 and 2 ID, there was no increase in implantation rate (IR) and live birth rate (LBR) after IVIG in comparison with patients with the same number of ID but without IVIG correction. After IVIG therapy decreased IR and LBR were restored in women with three or more immune deviations. Multiple immune deviations indicate IVF patients who may benefit from IVIG therapy. IVIG seems to convert "unfavorable" immune phenotype to "favorable" one.

Favorable immune phenotype predicts successful implantation and pregnancy.

Immune markers that may predict IVF failure and successful implantation and pregnancy were studied. Favorable immune parameters were selected based on 90% of data of women who got pregnant and had uneventful pregnancy course and outcome in present IVF cycle. Immune phenotype and NK cell activity of peripheral blood of 123 women with multiple IVF failure were studied by flow cytometry. Some parameters that were out of favorable borders (elevated expression of CD56, CD158a in T lymphocytes, decreased levels of CD4 T lymphocytes, up-regulated expression of HLA DR in CD8+ T cells and NK cells, elevated number of NK cells and increased NK cytotoxicity, increased and decreased expression of CD158a and CD8 in NK cells) were considered to be immune deviations (ID) potentially predictive for IVF failure. In women with 0-1 ID implantation rate (IR) was 50.9% (27/53), with two ID - 42.8% (12/28), with three and more ID - 21.4% (9/42). IR in group with three ID was lower than in group with 0-1 ID (p<0.01, OR=3.8, CI: 1.52-9.48) and in group with two ID (p<0.05). Live birth rate (LBR) in women with 0-1 ID was 33.9%, with two ID - 28.5%, with three and more ID - 9.5%. LBR in group with three ID was lower than in group with 0-1 ID (p<0.01, OR=4.8, CI: 1.52-15.8) and in group with two ID (p<0.05). The absence or single ID seems to be more favorable for successful IVF program. Combination of ID may predict implantation failure to a greater degree than isolated ID. Multiple immune deviations form unfavorable "immune phenotype" for implantation and pregnancy development.

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.

Preimplantation factor inhibits circulating natural killer cell cytotoxicity and reduces CD69 expression: implications for recurrent pregnancy loss therapy.

Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo development in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25ng/ml (37% and 42%) compared with PIFscr (18%; P<0.001), regardless of the proportion of peripheral-blood NKCD56+ cells to lymphocytes. Pre-incubation of blood from infertile patients with sPIF for 24h decreased NKCD69+ expression versus incubatino with PIFscr (P<0.05). In conclusion, sPIF inhibits NK cell cytotoxicity by reducing NKCD69 expression, suggesting a significant role in RPL patients. There is a continuous search to identify safe and effective agents to counteract recurrent pregnancy loss (RPL). Preimplantation factor (PIF) secreted by the embryo at the 2-cell stage is present throughout viable pregnancy but absent in nonviable pregnancy. Its immunomodulatory (not suppressive) effects promote embryo acceptance and maintenance by mother/host, control inflammation, facilitate uterine environment and placental embedding. Synthetic PIF (sPIF) was used to complete PIF's role as a targeted, safe treatment for immune-based RPL. Previous reports showed sPIF's significant protective systemic effect against maternal factors present in RPL serum. Herein is examined sPIF's ability to inhibit the local protective toxicity induced by natural killer (NK) immune cells in a representative number of RPL patients. When elevated in blood, NK cells are associated with RPL. Low-dose physiological sPIF was highly effective to inhibit NK cell toxicity. Side-by-side comparison showed that sPIF is equally effective at a lower dose than intravenous gamma immunoglobulin or Intralipid treatment currently used. The sPIF effect on NK cells was targeted, indicating specific action. Overall, sPIF may represent a safe, effective and nontoxic immune-based therapy against RPL.

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay.

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.

Elevated NK cell cytotoxicity, CD158a expression in NK cells and activated T lymphocytes in peripheral blood of women with IVF failures.

PROBLEM: The aim of this study was to evaluate the role of elevated natural killer cytotoxicity (NKc) in women with multiple implantation failures (IF) in vitro fertilization-embryo transfer (IVF-ET) cycles. METHODS OF STUDY: Seventy-nine antiphospholipid antibodies-negative women with IF including 33 women with elevated NKc were selected for investigation. K-562 cell line was used to evaluate NKc. Lymphocyte subsets, intracellular cytokines [interferon (IFN)-gamma, interleukin (IL)-4, tumour necrosis factor, IL-10], expression of activating markers [CD69, human leukocyte antigen (HLA)-DR], CD8, KIR (CD158a), CD95, and chemokine receptors (CXCR3, CCR4) were estimated by flow cytometry. RESULTS: In women with IF, levels of NKc were higher than in IVF successful women. IF was associated with higher expression of CD8, CD158a, and HLA-DR in NK cells, activating markers in T lymphocytes, and lower levels of CCR4+ and IL-4+ T lymphocyte subsets. Predictive value of single elevated NKc for IVF success was 0.85, but addition of two other abnormal parameters resulted in its decrease to <0.39. CONCLUSIONS: Elevated NKc is negative factor, though not critical for implantation in IVF cycles. Immune mechanism of IVF failure includes not only elevated NKc but also some other factors, such as elevated expression of CD8 and CD158a on NK cells, T lymphocyte activation, and diminished T helper 2 parameters.

Up-regulation of interferon-gamma production by reduced glutathione, anthocyane and L-cysteine treatment in children with allergic asthma and recurrent respiratory diseases.

Negative correlation between serum IgE levels and production of IFN-gamma by lymphocytes and positive correlation between serum IgE levels and production of IL-4 by lymphocytes was detected in 12 children with allergic asthma and recurrent respiratory diseases. Deficiency of reduced glutathione in whole blood and some disorders in phagocytic and oxidative burst activity of monocytes were observed in these children. Use of reduced glutathione, L-cysteine and anthocyane (Recancostat, Clear Vision, Switzerland) resulted in elevation of IFN-gamma production, lymphocyte response to mitogens, NK cell activity, increase in percentage of naive CD4(+) T lymphocytes (refreshment effect) and improvement of clinical status. Positive clinical results were lasted during 6 months.