Elevated Levels of Serum Vascular Endothelial Growth Factor-A Are Not Related to NK Cell Parameters in Recurrent IVF Failure.

BACKGROUND: Vascular Endothelial Growth Factor and NK cells have an interrelated role in angiogenesis that is critical for placentation and success of in vitro fertilization. An attempt was made to assess a possible relationship between the two in this study. METHODS: A case control study was performed comparing the serum levels of VEGF-A and its receptor VEGF-R1 with levels of NK cells, activated NK cells and NK cytotoxicity in 62 women with Repeated Implantation Failure (RIF). The healthy control group consisted of 72 women of similar age, without known issues in achieving pregnancy or evidence of autoimmunity. Levels of VEGF-A and VEGF-R1 were quantified by ELISA methods with standard curve interpolation. NK cell subsets were determined with flow cytometry using fluorescent-tagged anti-CD56, anti-CD16, anti-CD3 and anti-CD69. NK cytotoxicity was performed by incubating peripheral blood mononuclear cells and K562 cultured cells with propidium iodide, steroid, intralipid and intravenous immunoglobulin, using previously described methods. Statistical analysis involved Mann-Whitney-U and Spearman's rank correlation testing with p-values defined as <0.05. RESULTS: It was found that VEGF-A levels were significantly raised in women with RIF compared to healthy controls (362.9 vs. 171.6 pg/ml, p<0.0001), with no difference in VEGF-R1 levels between groups (1499 vs. 1202 pg/ml, p=0.4082). There was no correlation between VEGF-A or VEGF-R1 and the absolute levels of circulating NK cells, CD69 activated NK cells or NK cytotoxicity. CONCLUSION: The absence of correlation between VEGF-A or VEGF-R1 and NK cells suggests VEGF secretion and regulation is independent of NK cell activity in RIF.

Serial estimation of Th1:th2 cytokines profile in women undergoing in-vitro fertilization-embryo transfer.

PROBLEM: To investigate changes in the ratio of T-cell subpopulations expressing intracellular T helper1 (Th1) and T helper 2 (Th2) cytokines in women with a history of recurrent failed implantation under going in-vitro fertilization (IVF)-embryo transfer. METHOD OF STUDY: Twenty-eight peripheral blood samples were obtained at two time points, from 14 women undergoing IVF treatment; eight women with a history of recurrent failed implantation, who did not get pregnant in the index IVF cycle and six who had one or more previous successful IVF pregnancy and who became pregnant in the index IVF cycle. The proportion of lymphocytes expressing interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin 4 (IL-4) and the Th1:Th2 ratios of IFN-gamma:IL-4, and TNF-alpha:IL-4 in T helper cells was measured by flow cytometry, in samples obtained before commencing IVF treatment and in samples obtained after ovarian stimulation (on the day of oocyte retrieval). RESULTS: In samples collected during oocyte retrieval, women with a history of recurrent failed implantation had a higher IFN-gamma:IL-4 and TNF-alpha:IL-4 ratio than the control group, (18.6+/-9.3 versus 6.47+/-1.68, P=0.009) and (39.1+/-15.7 versus 11.53+/-3.76, P=0.001) respectively. In women with a history of recurrent failed implantation the ratio of IFN-gamma:IL-4 and TNF-alpha:IL-4 at oocyte retrieval was higher than pre-treatment ratios (18.6+/-9.3 versus 12.01+/-9.8, P=0.018) and 39.10+/-15.7 versus 18.66+/-11.42, P=0.010) respectively, showing a Th1 bias. In women with a successful IVF the converse was true; the ratio at oocyte retrieval was significantly lower than pre-treatment ratios (6.47+/-1.68 versus 9.37+/-6.8, P=0.035) and 11.53+/-3.76 versus 18.60+/-12.9, P=0.027) respectively, representing a Th2 bias. CONCLUSION: Women with a history of unexplained recurrent failed IVF treatment have a Th1 bias and this polarization is more enhanced following hormonal manipulations during IVF treatment. Comparing pre-treatment ratios of IFN-gamma:IL-4 and TNF-alpha:IL-4 to ratios obtained at oocyte retrieval may be clinically useful. Women with recurrent failed IVF have increasing ratios.

Prednisolone suppresses NK cell cytotoxicity in vitro in women with a history of infertility and elevated NK cell cytotoxicity.

PROBLEM: To evaluate the effect of prednisolone on NK cell cytotoxicity in vitro environment and also to compare the effect of prednisolone versus immunoglobulin-G (IVIG) on NK cell cytotoxicity using in vitro co-culture with K562 cells. METHOD OF STUDY: The following is a prospective observational study, between August 2006 and February 2007, was carried out on blood samples from 110 patients with a history of recurrent miscarriage or recurrent failed implantation. Peripheral blood mononuclear cells containing NK cells were isolated and co-cultured with target cell K562 in three different effector-to-target (E:T) ratios of 50:1, 25:1 and 12.5:1. Prednisolone or IVIG was then added to the tube with E:T ratio of 50:1 to assess suppressive effect. The percentage killing was recorded and statistical analysis performed using Student's t-test. RESULTS: In the experiments with an E:T ratio of 50:1 without prednisolone or IVIG in the co-culture, the mean target cell killing percentage was 26.4%. In cultures using the same E:T ratio, this killing percentage was significantly reduced in the presence of IVIG (9.9%) or prednisolone (13.6%), (P<0.001 in both analyses). On comparing the reduction in killing percentage of target cells by prednisolone versus IVIG, a slightly lower reduction in the prednisolone co-culture was noted but this was not statistically significant (P>0.05). CONCLUSION: The results of this study show that prednisolone is able to suppress the cytolytic activity of the NK cell. Prednisolone and IVIG are almost equally effective in suppressing in vitro NK cell cytolytic activity.

The relationship of systemic TNF-alpha and IFN-gamma with IVF treatment outcome and peripheral blood NK cells.

BACKGROUND: To evaluate the association of serum tumour necrotic factor (TNF)-alpha and interferon (IFN)-gamma levels with IVF treatment outcome and peripheral blood NK cells. METHODS: Prospective observational study of 126 randomly selected women who underwent IVF treatment. The serum levels of TNF-alpha and IFN-gamma were determined by multiplex suspension beads array system. RESULTS: There were no significant differences with regard to the systemic TNF-alpha and IFN-gamma levels between the pregnant (n = 51, TNF-alpha: 53.5 pg/mL; IFN-gamma: 4.6 pg/mL) and not pregnant (n = 75, TNF-alpha: 63.0; IFN-gamma: 7.5) women after IVF treatment. For those women with a positive pregnancy after IVF treatment, the systemic TNF-alpha and IFN-gamma levels were higher in those women who miscarried (n = 13, TNF-alpha: 67.4; IFN-gamma: 9.1) when compared with those who had a live birth (n = 38, TNF-alpha: 48.7; IFN-gamma: 1.4), however this difference was not statistically significant. Interestingly, the systemic TNF-alpha and IFN-gamma levels were significantly higher in women who had a higher level of activated (CD69(+)) NK cells (n = 39, TNF-alpha: 86.8; IFN-gamma: 4.7) when compared with women who had a low level of activated NK cells (n = 87, TNF-alpha: 46.9; IFN-gamma: 1.7 P = 0.028 and 0.045 respectively). CONCLUSION: The systemic levels of TNF-alpha and IFN-gamma have no association with implantation rate or miscarriage rate in women undergoing IVF treatment. However, high levels of TNF-alpha and IFN-gamma are associated with elevated levels of activated NK cells and this may subsequently exert a negative impact on reproduction.

The effect of serum concentration of leukaemia inhibitory factor on in vitro fertilization treatment outcome.

PROBLEM: To evaluate the association of peripheral leukaemia inhibitory factor (LIF) levels on implantation and miscarriage rates after in vitro fertilization (IVF) treatment. METHODS: Prospective observational study of 120 randomly selected women who underwent IVF treatment. The concentration of LIF in serum was determined by enzyme-linked immunosorbent assay. RESULTS: There was no significant differences with regard to the systemic mean LIF concentration between the pregnant (42 patients, LIF: 11.55 pg/mL +/- 5.3 S.D.) and non-pregnant (66 patients, LIF: 13.47 pg/mL +/- 5.1 S.D.) women after IVF treatment. Likewise, for those women who have positive pregnancy after IVF treatment, the systemic mean LIF levels were not significantly different between women who have an ongoing pregnancy (34 ongoing pregnancy, LIF: 11.26 pg/mL +/- 5.2 S.D.) and those who had miscarriage (eight miscarriage, LIF: 12.78 pg/mL +/- 5.6 S.D.). CONCLUSION: The systemic levels of LIF concentration have no association with implantation rate or miscarriage rate in women undergoing IVF treatment. Measuring serum LIF concentration prior to embryo transfer in IVF treatment has no predictive value of implantation rate or miscarriage rate.