Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.

Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology.

The aim of this study was to investigate the functional status and immunophenotypic characteristics of natural killer (NK) cells in women who suffer recurrent spontaneous abortions (RSA) or have infertility of unknown aetiology. Peripheral blood mononuclear cells (PBMC) were obtained from 40 study patients and 13 normal healthy multiparous controls. NK cells were identified using anti-CD56 and anti-CD16 monoclonal antibodies (mAb). The expression of CD69, CD25, CD122, CD30, CD154, CD128 and CD94 on NK cells was detected using specific mAb and analysed by flow cytometry. CD69 expression on NK cells after ED(27) human trophoblast cell line co-culture with PBMC was also investigated. A significant increase in CD69 expression on CD56(+) NK cells was demonstrated in women with RSA (P < 0.005) and infertility (P < 0.05) as compared with that of normal controls. Conversely, CD94 expression was significantly decreased in women with RSA (P < 0.005) and infertility (P < 0.05) in comparison with that of controls. Increased CD69 expression on NK cells was induced after 24 h co-culture with ED(27). In conclusion, peripheral blood NK cells of women with RSA and infertility of unknown aetiology have higher proportions of activated NK cells in vivo. Unbalanced CD69 and CD94 expression may explain the underlying pathology.

Immunoglobulin G infusion treatment for women with recurrent spontaneous abortions and elevated CD56+ natural killer cells.

We aimed to investigate the clinical effect of intravenous immunoglobulin G (IVIg) treatment in recurrent aborters with elevated peripheral blood CD56+ NK cell levels while on lymphocyte immunization, anticoagulation and prednisone treatment, with respect to subsequent live birth and reproductive outcome. Thirty-three women with recurrent abortions achieved alloimmune recognition after lymphocyte immunizations. All had autoimmune abnormalities and received preconception anticoagulation and prednisone treatment. At the time of positive pregnancy testing, 18 women with normal NK cell levels (<12%) and 6 with elevated NK cell levels (>12%) continued anticoagulation and prednisone treatment, and 9 with elevated NK cell level initiated additional IVIg treatment. The live birth rates of women with elevated NK cell level (>12%) who initiated post-conception IVIg treatment in addition to anticoagulation and prednisone (100.0%), women with normal NK cell levels (<12%) who continued anticoagulation and prednisone (83.3%) and women with elevated NK cell level (>12%) who continued anticoagulation and prednisone (33.3%) are significantly different (P=0.0065). Prevalence of intrauterine growth retardation and preterm delivery among 3 study groups were not different. In conclusion, post-conception IVIg treatment significantly improves reproductive outcome in women with elevated CD56+ NK cells with pregnancy who received preconception lymphocyte immunization, anticoagulation and prednisone treatment.

Immunopathology of the implantation site utilizing monoclonal antibodies to natural killer cells in women with recurrent pregnancy losses.

PROBLEM: Placental lesions of 71 women with documented recurrent spontaneous abortions of unknown etiology were evaluated using immunohistochemical staining. METHOD OF STUDY: Placental tissue blocks (less than 12 weeks gestation) from prior pregnancy losses were obtained, recut, and analyzed utilizing monoclonal antibody to identify the trophoblast (cytokeratin 8/18) and natural killer (NK) cells (CD57) at the implantation site. The following features were evaluated: trophoblast invasion pattern; syncytium formation; vasculitis and thromboembolism of decidual vessels; decidual inflammation; decidual necrosis; fibrin deposition at the decidual necrosis site; mononuclear-cell infiltration in villi and intervillous space; perivillous fibrin deposition; trophoblast morphology; and quantitation of CD57+ NK cells within the decidual tissue near the implantation site. Controls consisted of 20 healthy women with no history of recurrent pregnancy losses, who had their pregnancies electively terminated. RESULTS: Of the women studied, 29.6% demonstrated elevated CD57+ NK cells at the implantation site (P = 0.030), 54.1% had inadequate cytotrophoblast invasion depth (P = 0.000), 44.1% demonstrated inadequate syncytium formation (P = 0.004), and 33.9% presented thromboembolism in decidual vessels (P = 0.025). CONCLUSION: Some women with recurrent spontaneous abortions demonstrate abnormal placental lesions at the implantation site. Immunopathologic evaluation of the placental implantation site that terminated in a spontaneous abortion may reveal the immunopathogenesis of previous pregnancy losses.

Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions.

PROBLEM: Natural Killer (NK) cell measurement and NK cytotoxicity are two measurements for assessing the cellular immune response. Both of the techniques have been reported to be prognostic for women with recurrent spontaneous abortion (RSA). We evaluated the two methods to determine the relationship of the two assays. Because both methods portend to evaluate the same process, the previous clinical data suggested that the methods evaluate the same phenomena. We undertook these studies to determine whether simple NK cell counts may be sufficient in the evaluation of NK activity in RSA. METHOD OF STUDY: The NK cell cytotoxicity at effector-to-target ratios of 50:1 and 25:1 was determined using a flow cytometric NK cell cytotoxicity assay. These values were then correlated with the percentages and absolute counts of three peripheral blood NK cell subsets. RESULTS: The data indicate that the flow cytometric assay is reproducible and precise and can be successfully used to evaluate patient samples. Linear regression analysis indicated a lack of correlation between peripheral blood NK cell cytotoxicity and percentages or absolute counts of CD56+CD16+, CD56+CD16- or CD3+CD56+ lymphocyte subsets (range of correlation coefficients, 0.1-0.3). CONCLUSIONS: NK cell cytotoxicity and peripheral blood NK cell values measure different aspects of NK cells and do not correlate. These data indicate that simple enumeration of NK cells may not be sufficient in the evaluation of NK cells in RSA.

Natural killer cell cytotoxicity and paternal lymphocyte immunization in women with recurrent spontaneous abortions.

PROBLEM: Natural killer (NK)-cell cytotoxicity in women undergoing lymphocyte immunization prior to and following treatment was investigated. METHOD OF STUDY: A cohort of 33 women with a history of two or more recurrent spontaneous abortions was prospectively studied. NK-cell cytotoxicity was determined at effector-to-target ratios of 50:1 and 25:1. Peripheral blood CD56+ NK-cell, CD19+ B-cell, CD19+/5+ B-1-cell, and CD3+ pan T-cell levels were studied by flow cytometry before and after lymphocyte immunization treatment. Maternal antipaternal T- and B-cell antibody levels were measured before and after lymphocyte immunization by flow cytometric analysis. Paternal lymphocyte immunizations were given two times with a 4-week interval. Post-lymphocyte immunization testing was done 4 weeks after the second lymphocyte immunization. The controls were 8 normal healthy women. NK assays were done twice with an interval of 8 weeks. RESULTS: NK-cell activity at effector-to-target ratios of 50:1 (P = 0.005) and 25:1 (P = 0.001) were significantly suppressed after lymphocyte immunization. CD3+ pan T-cell levels after lymphocyte immunization were significantly increased compared with levels before lymphocyte immunization (P = 0.008). CD56+ NK-cell levels were significantly suppressed after lymphocyte immunization (P = 0.016). There was no correlation between changes in NK cytotoxicity and differences in antipaternal lymphocyte antibody levels before or after lymphocyte immunization. CONCLUSION: Lymphocyte immunization suppresses NK-cell cytotoxicity and CD56+ NK-cell levels and increases the peripheral blood CD3+ T-cell population in women with recurrent spontaneous abortions.

Effect of intravenous immunoglobulin G on natural killer cell cytotoxicity in vitro in women with recurrent spontaneous abortion.

Intravenous immunoglobulin (IVIg) has been used to treat women with recurrent spontaneous abortion (RSA), particularly for women with elevated natural killer (NK) cells. We investigated the effect of IVIg on peripheral blood NK cell activity in vitro in women with RSA. 51Cr-release assays using K562 in the presence of varying concentrations of IVIg were performed using PBL from 16 women with RSA. Antibody dependent cellular cytotoxicity (ADCC) was evaluated using Daudi cells. Effectors and targets were preincubated with IVIg. Binding of IVIg to K562 and Daudi was evaluated by flow cytometry. The effect of K562 absorbed IVIg on NK activity was compared to that of non-absorbed IVIg. NK cytotoxicity and ADCC in the presence of F(ab')2 fragments were compared with those in the presence of intact IVIg. IVIg produced a significant, dose dependent inhibition of NK activity in vitro. Inhibition of NK activity occurred when effectors but not targets were preincubated with IVIg. IVIg binds to K562 and Daudi. IVIg increased ADCC when targets but not effectors were incubated with IVIg. K562 absorbed IVIg produced more inhibition of NK cytotoxicity than non-absorbed IVIg. Suppression of NK cytotoxicity by F(ab')2 was as effective as that of IVIg. However, F(ab')2 did not increase ADCC. IVIg effectively reduces peripheral blood NK cytotoxicity in vitro. Inhibition of NK cytotoxicity is mediated at the effector cell level through the antigen binding portion of the immunoglobulins. Women with RSA and elevated NK cells may benefit from IVIg treatment.

Elevated peripheral blood natural killer cells are effectively downregulated by immunoglobulin G infusion in women with recurrent spontaneous abortions.

PROBLEM: We investigated the hypothesis that elevated peripheral blood natural killer cells (NK) are decreased by immunoglobulin G infusion (IVIg) therapy in women with recurrent spontaneous abortions (RSA) and elevated NK cells. METHODS: Seventy-three women with RSA and elevated NK cells received IVIg therapy (400 mg/Kg/day for 3 days ever 4 wks) and anticoagulation treatment. Peripheral blood immunophenotype assay by flow cytometry was done prospectively prior to and 7 days after first IVIg therapy, every 2 wks until 20 wks gestation and then monthly. Controls were 95 women with RSA and normal NK cells who received anticoagulation treatment. RESULTS: (1) 86.3% of women with elevated NK cells who received the IVIg and anticoagulation therapy had a successful pregnancy outcome; (2) Peripheral blood CD56+ NK cells and CD56+/16+ NK cells were significantly suppressed 7 days post IVIg infusion (P < 0.0005); (3) Pre-IVIg infusion levels of other lymphocyte subsets were not different as compared with those of 7 days post-IVIg therapy; (4) Women who delivered a liveborn infant with IVIg therapy demonstrated downregulation of peripheral blood NK cells (CD56+, CD56+/16+) during early pregnancy when compared to women who miscarried the index pregnancy (P < 0.05); (5) Women with normal NK cells who miscarried while on anticoagulation therapy demonstrated significantly elevated CD56+ NK cells during early pregnancy as compared with that of women who delivered a liveborn infant (P < 0.05); (6) CD19+ B cells were significantly downregulated during pregnancy in women with anticoagulation and IVIg therapy when compared to women with anticoagulation therapy (P < 0.05). CONCLUSION: Downregulation of NK cells in women with RSA is associated with a favorable pregnancy outcome. Peripheral blood NK cells (CD56+, CD56+/16+) are effectively suppressed after IVIg therapy. Women with RSA and high NK cells benefit from IVIg therapy and experience suppression of CD56+ and CD56+/16+ NK cells.

Intravenous immunoglobulin inhibits natural killer cell activity in vivo in women with recurrent spontaneous abortion.

We previously reported elevation of natural killer (NK) cells in women with recurrent spontaneous abortion (RSA) of immune etiology. In this study, we investigated the effect of intravenous immunoglobulin G (IVIg) on peripheral blood NK activity in vivo in women with RSA. Blood was drawn prior to and 7-11 days after IVIg therapy in eight women with RSA. NK activity was measured using K562 as target cells for 51Cr-release assays. Serum IgG concentrations were also measured. All received 400 mg/kg/day of IVIg for 3 consecutive days. 1) Seven of eight women became pregnant. Five delivered a live born infant. Three out of five women (60%) who delivered a live born infant showed a significant inhibition of NK cytotoxicity post IVIg and the rest did not show any changes; 2) NK cytotoxicity was significantly increased in a woman who miscarried again; 3) A woman who miscarried a chromosomally abnormal fetus showed a significant inhibition of NK cytotoxicity after IVIg; and 4) Serum IgG concentration increased significantly from 9.3 +/- 3.0 mg/ml to 23.5 +/- 5.1 mg/ml post IVIg therapy. IVIg effectively inhibits peripheral blood NK activity in vivo. These results are consistent with our previous finding showing that IVIg inhibits NK cell activity in vitro. Women with RSA and elevated NK cells may benefit from IVIg treatment.

Immunophenotypic profiles of peripheral blood lymphocytes in women with recurrent pregnancy losses and in infertile women with multiple failed in vitro fertilization cycles.

In summary (1) Nonpregnant women with RSAs of unknown etiology have higher levels of CD56+ lymphocytes when compared to normal controls; (2) The levels of CD19+, CD56+, and CD56+/CD16+ PBL of pregnant women with RSA are significantly higher than those of multiparous pregnant normal controls; (3) Women with autoantibodies to phospholipids have significantly higher levels of elevated CD56+ and CD56+/CD16+ lymphocytes when compared to women without antiphospholipid antibodies; (4) Women with autoantibodies to nuclear components demonstrate higher numbers of CD19+/CD5+ cells compared to women without autoantibodies to nuclear components; (5) Idiopathic infertile women with multiple prior IVF failures demonstrate significantly higher levels of CD56+ pBL than normal fertile controls and the conception rate is much higher in those with CD56+ levels less than 12%; (6) Elevations of CD56+ lymphocytes to over 18% during a pregnancy is a good prognostic indicator of impending pregnancy loss. We have not seen a liveborn infant in women with levels of 18% or higher without IVIg therapy; and (7) Infertile and RSA women who fail alloimmune and autoimmune therapy have significant alterations in cellular and humoral immunity involving NK cells and CD19+/CD5+ B cells.

Impact of age on reproductive outcome in women with recurrent spontaneous abortions and infertility of immune etiology.

The objective of this paper is to determine whether age has any impact on conception rate, pregnancy outcome, or autoimmune status in women with recurrent spontaneous abortions (RSA) and infertility of immune etiology. One hundred twenty-four women with 3 or more RSA and 36 women with unexplained infertility were prospectively studied. Maternal antipaternal lymphocyte antibodies and autoantibodies to phospholipids and nuclear antigens were tested. All achieved an adequate alloimmune recognition after lymphocyte immunization and followed for 1 year with optimal preconception autoimmune treatment. Conception rate and pregnancy outcome were prospectively studied. 1) 10.1% of women with RSA and 30.6% of women with infertility failed to achieve a pregnancy after 1 year of trial (P = 0.0084); 2) in women with RSA, the number of previous fetal death after 28 weeks of gestation was significantly higher in women who failed to achieve a pregnancy within 1 year when compared to women who became pregnant (P = 0.0296). Conception rate was not different with advancing age; 3) women with infertility demonstrated significantly higher incidence of anti-phosphatidylethanolamine antibody when compared to women with RSA (P = 0.052); 4) in women with infertility, those who failed to achieve a pregnancy were significantly older (P = 0.0012) and demonstrated a higher incidence of autoantibodies to phosphatidic acid than women with infertility who became pregnant (P = 0.0339); 5) the subsequent spontaneous abortion rate while on optimal immune therapy was the same in the women with RSA (39.3%) and women with infertility (32%). Spontaneous abortion rate of women with RSA or infertility was not different among four age groups; 6) the presence of anticardiolipin antibody (P = 0.0055) and higher gravidity (P = 0.0354) correlated significantly with pregnancy failure in women with a history of infertility. The prevalence of auto-antibodies to phospholipids and nuclear components was not different among four age groups in women with RSA or infertility. Age does not affect pregnancy outcome or conception rate in women with RSA. In women with infertility of immune etiology, conception rate was significantly reduced over age 40 although pregnancy outcome was no different with advanced age.

Expression of membrane form of the pregnancy associated protein TJ6 on decidual lymphocytes in the first trimester of pregnancy.

TJ6, a newly described protein produced locally in the uterine decidua during pregnancy, may be involved in maintaining a unique immunological environment at the maternal-fetal interface. The aim of this study was to determine whether TJ6 is expressed as membrane form on decidual lymphocytes (DL), to define the phenotypes of TJ6m (membrane form TJ6) expressing cells and to analyze the fluorescence intensity of TJ6m expression. Peripheral blood lymphocytes (PBL) and DL were obtained from first trimester pregnancies undergoing elective termination and immunophenotyped for TJ6m and other cell surface antigens (CD3, CD8, CD19, CD56, CD16) by flow cytometry. This is the first study showing that TJ6 molecules are present on decidual lymphocytes in human pregnancy. TJ6m expression on PBL was not different from that of DL. However, a significantly higher percentage of double positive (TJ6m+CD3+, TJ6m+,CD8+,TJ6m+CD19+) cells were found in PBL when compared to DL. The average fluorescence intensity (AFI) for the TJ6m marker among cells with CD8+, CD19+ and CD56+ double positive was significantly higher in DL as compared with those of PBL. The AFI for granularity of double positive DL was significantly higher than observed in PBL.

Analysis of health insurance cover for reproductive immunology.

The objective of this study was to document the current state of health insurance cover for reproductive immunology in the USA. A survey of couples who had given birth to a child within the last 2 years while they were being treated with lymphocyte immunotherapy at the Reproductive Medicine Clinic of the Finch University of Health Sciences/The Chicago Medical School (FUHS/CMS), North Chicago, IL, USA, produced 61 completed questionnaires from couples in 16 states, representing a response rate of 55%. The Reproductive Medicine Clinic at the FUHS/CMS is a major centre that treats couples with reproductive immunological problems. These couples had experienced repeated pregnancy losses that were diagnosed as immunological in nature. This prospective study documents the insurance reimbursement of couples receiving lymphocyte immunotherapy who subsequently became parents. The findings indicate that 80% of couples had insurance claims initially denied for reproductive immunotherapy-related services. The most common reason given for a denied claim was that the treatment was experimental. Couples took further action and almost all received cover. Cover for immunotherapy-related services averaged 65% of the cost. The percentage of expenses covered by the insurance was quite variable. The survey indicates that insurance cover is provided if patients are willing to take action.

Up-regulated expression of CD56+, CD56+/CD16+, and CD19+ cells in peripheral blood lymphocytes in pregnant women with recurrent pregnancy losses.

PROBLEM: To analyze immunophenotypic profiles of peripheral blood and humoral autoimmune responses in women with a history of recurrent spontaneous abortions (RSA). METHOD: Peripheral blood lymphocyte subsets by flow cytometry and autoantibodies to phospholipids and nuclear components by ELISA were measured in nonpregnant and pregnant women with RSA of unknown etiology. Thirty-five pregnant and eighty-one nonpregnant women with RSA were studied. Seventeen nonpregnant and twenty-two pregnant normal controls were included. RESULTS: Natural killer (NK) cells (CD56+) were significantly elevated in nonpregnant women with RSA as compared with nonpregnant controls. Pregnant women with RSA demonstrated significantly increased NK (CD56+, CD56+/CD16+) and B cells (CD19+) as compared with pregnant controls. Women who miscarried the index pregnancy demonstrated significantly lower CD3+ cells in comparison with normal controls. Women with RSA and antiphospholipid antibodies showed significantly elevated NK cells when compared with women without antiphospholipid antibodies. Women with autoantibodies to nuclear components demonstrated significantly elevated CD19+/CD5+ cells when compared to women without autoantibodies to nuclear components. CONCLUSIONS: Women with RSA demonstrate an abnormal cellular immune response by increasing peripheral natural killer cells and B cells as compared with normal controls.

Intravenous immunoglobulin infusion therapy in women with recurrent spontaneous abortions of immune etiologies.

We have investigated clinical effectiveness of intravenous immunoglobulin G infusion (IVIg) on antiphospholipid antibody titers in five women with evidence of antiphospholipid antibody-associated recurrent spontaneous abortions and one with antinuclear antibody who became refractory to conventional autoimmune treatment during pregnancy and experienced pregnancy complications. Three women developed intrauterine growth retardation and three had complicated twin pregnancies with rising autoantibody titers. Antiphospholipid antibody and antinuclear antibody titers were tested pre and 2 weeks after each IVIg infusion. We report that: (i) IgG antiphospholipid antibody titers were significantly suppressed after each IVIg infusion (P < 0.05); (ii) IgM antiphospholipid antibody titers were also significantly suppressed after each IVIg infusion (P < 0.0001); (iii) decreased titers of autoantibodies paralleled increased levels of maternal IgG which lasted for at least 30 days; the autoantibodies showed a definite rise again prior to the next infusion; (iv) antinuclear antibody titers were effectively suppressed; and (v) rising autoantibody titers combined clinical manifestation of intrauterine growth retardation and women with complicated twin pregnancies. We conclude that IVIg infusion effectively suppresses IgM and IgG autoantibodies to phospholipids and antinuclear antibody in autoimmune women with a history of recurrent spontaneous abortions and refractory to conventional anticoagulation or immunosuppressive treatment.

Decidual-trophoblast interactions: decidual lymphoid cell populations in basal and parietal decidua.

Immunohistochemical analysis of tissue specimens from human pregnancy decidua basalis in contact with invasive trophoblast of chorion frondosum and decidua parietalis in contact with non-invasive chorion laeve do not differ in the frequency of lymphoid cells of the following phenotypes (CD2, CD4, CD8, CD14, CD21 and gamma/delta TCR). A practical implication of this observation is that the collection of lymphoid cells from whole decidua by curettage for functional studies is justified.

Autoantibodies to phospholipids and nuclear antigens in non-pregnant and pregnant Colombian women with recurrent spontaneous abortions.

Autoantibodies to negatively charged phospholipids have been reported to be associated with thrombotic events, thrombocytopenia and adverse pregnancy outcome, such as intrauterine growth retardation and recurrent spontaneous abortions (RSAs). In this study, autoantibodies to 6 phospholipid antigens and antinuclear antibody (ANA) were tested in Colombian women with a history of RSAs. Sixty-eight non-pregnant and 25 pregnant women with a history of RSAs comprised the study group. Twenty-five non-pregnant normal healthy women and thirty-one normal pregnant women served as controls. The non-pregnant women with RSAs showed a higher incidence of autoantibodies to cardiolipin (23% positive) as compared with non-pregnant normal controls (0% positive; P < 0.005). The incidence of autoantibodies to cardiolipin (28%; P < 0.005), phosphatidylethanolamine (16%; P < 0.005), phosphatidylserine (16%; P < 0.05), phosphatidylglycerol (16%; P < 0.05), phosphatidic acid (16%; P < 0.01) and phosphatidylinositol (20%; P < 0.01), in the pregnant women with RSAs was significantly higher than that of normal pregnant controls. There was no difference in the incidence of ANA in either group. In conclusion, women with a history of RSAs have a higher incidence of autoantibodies to phospholipids when compared to pregnant and non-pregnant normal controls. Autoimmune serological work-up is indicated during pregnancy in women with a history of RSAs.