1 Reproductive Medicine, Department of Obstetrics and Gynecology and 2 Department of Microbiology and Immunology, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, N. Chicago, IL 60064, USA
3 To whom correspondence should be addressed. e-mail: Kwakj{at}finchcms.edu
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Abstract |
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Key words: implantation failures/IVF failures/recurrent pregnancy losses/Th1/Th2 cytokines
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Introduction |
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Recently, significantly higher serum levels of Th2 cytokines, IL-6 and IL-10, were detected in normal pregnancy compared with unexplained recurrent pregnancy losses and significantly higher serum levels of the Th1 cytokine, IFN-, were present in women with recurrent pregnancy losses compared with normal pregnancy (Raghupathy et al., 1999
). These results suggested the notion that women with normal pregnancy have a Th2 bias, while women with a history of recurrent pregnancy losses have a bias toward Th1-type reactivity.
In pregnant mice, the injection of each Th1 cytokine, such as IFN-, TNF-
and IL-2, or co-administration of those significantly increased fetal resorption (Chaouat et al., 1990
; Clark et al., 1998
). It is suggested that Th1 cytokines trigger thrombotic/inflammatory processes at the maternal utero-placental blood vessels by activation of vascular endothelial cell procoagulant (Clark et al., 1998
). By contrast, Th2 cytokines inhibit Th1-induced tissue factor production by monocytes (Del Prete et al., 1995
).
Recurrent spontaneous abortion (RSA) is a common complication of pregnancy that may affect as many as 2% of women in reproductive age (Mills et al., 1988; Coulam, 1991
). Although genetic, anatomic and hormonal causes have been implicated in the aetiology of RSA (Stray-Pedersen and Stray-Pederson, 1984
; Carp et al., 1990
), >60% of cases remain unexplained. Various immunological abnormalities have been reported in women with RSA of unknown aetiologies including autoimmune abnormalities such as positive antiphospholipid antibodies, anti-nuclear antibodies, anti-thyroglobulin antibodies and anti-microsomal antibodies, and increased cellular immunity such as elevated natural killer (NK) cell levels and NK cytotoxicity (Kwak et al., 1995
; Ruiz et al., 1996
). Interestingly, these immunological abnormalities also occur in infertile women who have implantation failures after multiple IVF cycles (Beer et al., 1996
; Coulam et al., 1997
). These immunological similarities between women with RSA and infertility due to implantation failures led us to speculate that the proclivity to Th1 cytokine responses by circulating T cells in women with RSA (Raghupathy et al., 2000
) may exist in women with infertility of implantation failures. Previous studies of cytokine synthesis in peripheral blood of women with normal pregnancies or recurrent spontaneous aborters (Hill et al., 1995
; Raghupathy et al., 1999
) were designed to measure the total secreted cytokines from mononuclear cells but failed to discriminate the lymphocyte subpopulations. Thus studies investigating Th1/Th2 immune regulation in women with RSA or infertility of implantation failures by specifically defining the intracellular cytokine expression of CD3+/CD4+ T helper cells have not been reported. In this study, we have directly tested the Th1/Th2 paradigm in women with a history of RSA and women with multiple (two or more) implantation failures after IVF cycles by measuring intracellular Th1 and Th2 cytokine expression in CD3+, CD3+/CD8 (considered equivalent to CD3+/CD4+) or CD3+/CD8+ cells, and their ratios in subsets of peripheral blood T lymphocytes using 4-colour flow cytometry.
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Materials and methods |
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Inclusion criteria for this study were: (i) fertile women with three or more RSA of unknown aetiology or (ii) infertile women with two or more implantation failures after IVF cycles, who had two or more embryos transferred per each IVF cycle, excluding donor oocyte cycles; (iii) not pregnant; (iv) no more than one live born infant; (v) age ranges 2545 years old; and (vi) no active disease including autoimmune disease. To investigate a possible medical condition, physical examination, past history review, review of system and blood tests were performed. Blood tests were comprehensive metabolic panel, complete blood count, thyroid function test [free thyroxine (T4) and thyroid-stimulating hormone], antinuclear antibody, antibodies to single-stranded DNA, double-stranded DNA and histone.
The recurrent abortion group included 26 women with three or more RSA of unknown aetiology with the same partner. Three had one child. All had pregnancy losses during the first trimester of gestation. None had active autoimmune disease or a history of autoimmune disease. No one with evidence of active autoimmune disease was included in this study. No apparent causes of recurrent abortion such as chromosomal, endocrine, anatomical, or infectious aetiologies were documented for previous pregnancy losses. None had infertility or received IVF cycles. Table I lists age, obstetric histories of RSA groups and normal fertile controls. Age distribution is comparable between women with RSA and normal fertile controls.
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Laboratory
Cell separation and staining
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Biotech, Sweden) density centrifugation, and prepared and stained as previously reported (Ng et al., 2002). The cytokine expression levels were undetectable without stimulation after 5 h of culture with monensin only (Pharmingen, USA). Therefore, cells were stimulated using 25 ng/ml phorbol myristate acetate (PMA) and 1 µmol/l ionomycin in the presence of 2 µmol/l monensin, which inhibits cytokine secretion. Cells were stained according to the manufacturers instruction with the Cytofix/Cytoperm kit (Pharmingen). To detect intracellular cytokines, 0.2 µg of monoclonal antibody/x106 cells for IL-4 and 0.5 µg of monoclonal antibody/x106 cells for IFN-
, TNF-
and IL-10 were used. Anti-cytokine antibodies were: [phycoerythrin (PE)-anti-human IFN-
, clone 4S.B3; PE-anti-human TNF-
, clone Mab11; PE-anti-human IL-4, clone 8D4-8; PE-anti-human IL-10, clone JES3-19F1; PE-mouse IgG1 isotype, clone MOPC-21; PE-rat IgG2 isotype, clone R35-95 (Pharmingen). Corresponding isotype controls were utilized for each antibody and for each patient.
It has been shown that following stimulation of lymphocytes with PMA and ionomycin, a rapid down-regulation of CD4 molecules on the surface of lymphocytes occurs. In our hands, a decrease of CD4 occurred as rapidly as 4 h after stimulation. Therefore, a negative gating strategy was used to measure intracellular cytokine expression in CD3+CD4+ cells. Cells are reacted with phycoerythrinTexas Red (ECD)-anti-CD3 (BeckmanCoulter, USA) and fluoroscein isothiocyanate (FITC)-anti-CD8 (clone T8; BeckmanCoulter) and cells that were CD3+ but not CD8+ were considered to be CD3+CD4+ cells (Rostaing et al., 1999). In our study, the percentages of Th2 cytokine (IL-4 and IL-10)-producing cells were relatively low. Although the same pattern has been reported previously (Tsuda and Yamasaki, 2000
), we verified IL-10 and IL-4 data by analysing isotype controls. Non-specific intracellular staining with isotype control antibodies was 0.19 ± 0.15% for the IL-10 expression study and 0.17 ± 0.13% for the IL-4 expression study.
Acquisition and analysis of flow cytometric data
The stained and fixed samples were analysed on a Coulter XL flow cytometer using XL software (Coulter Corp., USA). Fluorescence from the FL1 (FITC), FL2 (PE), FL3 (ECD) and FL4 [phycoerythrin cyanin (PC5)] channels were used to measure cell surface and intracellular fluorescence as previously reported (Ng et al., 2002). The number of events acquired for each sample was 40 000. A region based on light scatter [forward angle light scatter (FALC) versus side scatter (SS)] was drawn around the major lymphocyte population. This population was used to obtain the gated fluorescence plot of CD3 versus CD8. Rectangular regions were set to include all the CD3+CD8+ bright cells and all the CD3+CD8 (CD3+/CD4+) bright cells obtained by negative gating strategy (Figure 1). All data are expressed as the percentage of cytokine-positive CD3+/CD8 or CD3+/CD8+ bright cells. A cell surface activation antigen (CD69, recognized by Pc5-anti-human CD69, clone TP.55.3; Beckman Coulter) was also used on these permeabilized and fixed cells to show the activation status of the CD3+/CD8 (CD3+/CD4+) and CD3+/CD8+ cells within the tight lymphocyte light scatter gate used for the analysis of intracellular cytokines.
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Unpaired two-tailed t-test was applied for comparisons of intracellular cytokine expression and proportion of T cell subsets between women with RSA and normal fertile controls. Th1/Th2 ratios in CD3+, CD3+/CD8 and CD3+/CD8+ cells of women with RSA were compared with those of normal fertile controls using unpaired two tailed t-test. If the population variances in the two groups were equal, the pooled-variance t-test was applied. If the population variances in the two groups are different, the separate variance t-test was applied. P 0.05 was considered significant.
For the comparison of intracellular cytokine expression, T cell subsets and the Th1/Th2 ratios among women with multiple implantation failures without SAB, with SAB and normal fertile controls, one-way analysis of variance with the Scheffé multiple comparison test was applied. The Scheffé multiple comparison test was applied to protect against identifying too many differences as significant. This test allows more stringent criteria for declaring differences significant than the usual t-test (Norusis, 1986).
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Results |
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To measure intracellular cytokines, resting lymphocytes must first be activated for 5 h with PMA and ionomycin. Less than 2% of CD3+ cells demonstrated CD69 expression without stimulation. For the confirmation of lymphocyte activation, CD69 expression on T cell subpopulations was measured in study and control subjects. A total of 88.2 ± 4.3% (mean ± SD) cells of women with RSA, 88.8 ± 4.1% cells of women with infertility of implantation failures without SAB, 88.6 ± 4.0% cells of women with infertility of implantation failures with SAB, and 87.4 ± 6.0% of normal fertile controls were activated as judged by the expression of the early activation antigen CD69 after 5 h incubation with PMA and ionomycin. The proportions of activated cells (CD69+) in study and control groups were not significantly different. These results suggested that lymphocyte stimulation with PMA and ionomycin was similar in study and control groups.
Intracellular cytokine expression
The proportion (%) of TNF--, IFN-
-, IL-4- and IL-10-producing CD3+, CD3+/CD8, CD3+/CD8+ cells in women with RSA, infertile women with multiple implantation failures without a history of SAB, with a history of SAB and normal fertile controls are listed in Table III. The proportion of IL-10-producing CD3+/CD8+ cells was significantly lower in women with RSA as compared with that of normal fertile controls (P = 0.013). The proportion of TNF-
-producing CD3+/CD8 cells was significantly different among women with implantation failures without SAB, with SAB and normal fertile controls (P = 0.011).
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Discussion |
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Recent advances in immunoassays such as commercially available standardized cell permeabilization reagents rather than saponin, antibodies to cytokines which are directly conjugated to give low background fluorescence, and the use of different combinations of monoclonal antibodies for 4-colour flow cytometry, have led to greater flexibility and more consistency in determining intracellular cytokine expression (North et al., 1996). In our study we investigated CD3+/CD4+ Th1 and Th2 cells by measuring CD3+/CD8 cells based on intracellular cytokine expression and also evaluated cytokine expression in CD3+/CD8+ cells in women with RSA or infertility of implantation failures. NK cells, which are CD3, were not included in the flow cytometric analysis. A concern may be raised that CD3+/CD8 cells are measured for CD3+/CD4+ cells. These two cell populations are probably identical, although by definition of expression they are not.
In our study, lymphocytes were stimulated with PMA and ionomycin in the presence of a protein transport inhibitor monensin to allow cytokines to accumulate in levels that were high enough to be detected by intracellular flow cytometric analysis. We have demonstrated the prevalence of Th1 immune responses over Th2 immune responses in specific T cell subpopulations based on expression of each cytokine. Previous in-vitro studies utilized trophoblast antigens to activate lymphocytes of women with a history of RSA and reported the presence of cytokines in the supernatant that was injurious to the developing conceptus or trophoblast cell lines (Ecker et al., 1993; Hill et al., 1995
). Our study suggests that altered Th1 immune responses can also be detected systemically as a result of an in-vivo challenge during pregnancy (RSA) or IVF cycles.
TNF- is supposed to suppress the growth of trophoblasts (Todt et al., 1996
), possibly by inducing apoptotic changes in these cells (Yui et al., 1994
). TNF-
is present on the proliferating tips of anchoring villi, invasive interstitial cytotrophoblasts, and endovascular trophoblasts which invade spiral arteries (Lea et al., 1997
). These findings suggest a role for TNF-
in early invasion of trophoblasts. However, a decrease in the release of TNF-
from PBMC upon the recognition of HLA-G was a consistent finding among normal women, recurrent aborters, and men (Maejima et al., 1997
). Perhaps the regulation of TNF-
synthesis may determine reproductive outcome. In our study, TNF-
expression in CD3+/CD8 cells from infertile women with implantation failures is significantly up-regulated as compared with that of normal controls. More importantly, the ratio of TNF-
to IL-4- or IL-10-expressing cells is persistently elevated in both women with RSA and infertility due to implantation failures with no history of SAB as compared with those of normal fertile controls, and infertile women with multiple implantation failures without SAB demonstrated the highest Th1/Th2 ratios, especially TNF-
related ratios. These findings are examples of the relationship between TNF-
, implantation and pregnancy outcome.
IL-10 is known to selectively suppress Th1-mediated cellular immunity by inhibiting the production of inflammatory cytokines such as IFN-, TNF-
and IL-1 (Mosmann and Moore, 1991
). Decreased production of IL-4 and IL-10 by decidual T cells of women with unexplained RSA when compared with decidual cells of women with normal pregnancy has been reported (Piccini et al., 1998
). Our study demonstrated a significant difference in IL-10 expression in activated peripheral blood CD3+/CD8+ cells in recurrent aborters.
In the mouse model, placental antigens from resorption-prone CBA/JxDBA/2 mating activate CD8+ T cells, which results in abortion (Raghupathy, 1997b). However, in-vivo injection of anti-CD8+ T cells into abortion-prone CBA/JxDBA/2 pregnancies either has no effect or boosts the abortion rate, dependent upon days of gestation (Chaouat and Menu, 1997
). CD8+ T cells have apparently opposing effects, which may in part be explained by the Th1/Th2 paradigm and the fact that CD8+ T cells can potentially belong to either phenotype. In our study, TNF-
/IL-10 ratios in CD3+/CD8+ T cells differed significantly between in women with infertility of implantation failure as compared with those of normal controls. Our collateral study using the same population of patients demonstrated significantly elevated activated NK cells (CD56+/CD69+) in peripheral blood (Ntrivalas et al., 2001
). The presence of activated NK cells in these women may be related to the activation status of CD3+/CD8+ cells, which have decreased Th2 cytokine production.
The underlying aetiology of Th1/Th2 polarization needs further investigation. A Th1 shift may be mediated by T cells or antigen-presenting cells that direct the differentiation of effector cells. From our study, it is interesting to notice that lymphocytes from women with implantation failures, who had never become pregnant and have had no chance to be exposed to trophoblasts antigens, or never had any history of pregnancy losses, demonstrated an increased Th1 shift in their cytokine expression. Therefore, it is plausible that increased synthesis of Th1 cytokines may be induced not only by trophoblast antigens, but also by antigen-non-specific cytokine/chemokine production in response to stress products of hormonally manipulated endometrium, hyperstimulated ovarian products or non-physiologically high levels of female sex hormones. In this study, incidence of endometriosis and tubal factor infertility in women without a history of SAB was higher than that of women with a history of SAB although the difference was not statistically significant. It is speculated that endometriosis and tubal inflammatory diseases may contribute to a Th1 shift in women with multiple implantation failures.
A question was raised as to whether women with implantation failure without a history of spontaneous pregnancy losses have a Th1 shift. In this study, women with implantation failures without a history of SAB were compared with women with implantation failures with a history of SAB and normal fertile controls. This study setting allowed us to investigate the impact of pregnancy losses and Th1 shift with regard to implantation failures. Interestingly, infertile women with multiple implantation failures without a history of SAB demonstrated significantly lower number of mature oocytes than infertile women with multiple implantation failures with SAB history in their previous IVF cycles. The possibilities that (i) pre-existing Th1 shift may cause low response to ovarian stimulation or (ii) higher dose FSH stimulation due to poor ovarian response may induce Th1 shift, should be explored in the future.
Since the introduction of IVF and embryo transfer technique, the implantation rate after IVF cycles has not markedly increased. Prenatal genetic diagnosis may explain this low implantation rate in part (Kahraman et al., 2000). However, our study also raises an important question for the role of Th1/Th2 immune responses in failure of embryonic implantation. Further study is needed to explore the relationship between T helper cell cytokine regulations, MHC complex, and reproductive outcome in women with altered Th1 immune responses.
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Submitted on May 3, 2001; resubmitted on August 16, 2002; accepted on December 9, 2002.