1 Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY 10021, USA
2 To whom correspondence should be addressed. e-mail: sdspando{at}med.cornell.edu)
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Abstract |
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Key words:
IFN-/IL-2 sR
/IVF/pregnancy outcome/TNF-
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Introduction |
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The processes involved in human pregnancy that allow an embryo, which is considered to be a semi-allograft, to develop inside a uterus without being rejected are still largely unclear (Brent, 1997). Recent evidence suggests that maternal tolerance is established at the feto-maternal interface, by factors deriving from the decidualized endometrium and from the trophoblast itself, and is maintained throughout gestation in physiological pregnancy (Bulla et al., 2003
). These factors, which are considered critical to bi-directional fetal immune tolerance and successful pregnancy outcome, include decidual immune cells and cytokines (Saito, 2001
). Thus, unexplained early pregnancy loss may result from alterations in the decidual immune population and/or disturbance of the delicate cytokine milieu (Dealtry et al., 2000
).
Cytokines are hormone-like polypeptides that interact with cell surface receptors to affect intracellular signals. In this respect, cytokines released at the feto-maternal interface have been proposed to play an important role in regulating embryo survival, controlling not only the maternal immune response but also angiogenesis and vascular remodelling (Ashkar et al., 2000; Bulla et al., 2003
). Recent studies originating from murine models have focused attention on the dichotomous T-helper response in reproduction, which states that pregnancy rejection is mediated by T-helper-1 (Th-1) cytokines, whereas a T-helper-2 (Th-2) cytokine response supports pregnancy (Wegmann et al., 1993
; Makhseed et al., 2001
).
Th-1 and Th-2 cells are the major subsets of fully differentiated CD4+ T cells, and their specific functions in immune responses correlate with their distinctive cytokine secretion (Raghupathy, 1997). In humans, Th-1 cells synthesize mainly interleukin-2 (IL-2), tumour necrosis factor-
(TNF-
) and interferon-
(IFN-
), whereas Th-2 cells produce IL-4, IL-5, IL-6 and IL-10 (Wu et al., 2001
). Th-1 cytokines are considered to be detrimental to pregnancy, via direct embryotoxic activity, or via damage to the placental trophoblast, or possibly by activating cells that are deleterious to the conceptus, whereas Th-2 cytokines may directly or indirectly contribute to the success of pregnancy by downregulating potential Th-1 reactivity (Raghupathy, 1997
; Makhseed et al., 2000
).
Since Th-1 cytokines may be detrimental to human embryo implantation and development, we prospectively measured the levels of these cytokines as early as 11 days after embryo transfer in consecutive IVF patients, in order to evaluate their predictive value in the outcome of IVF pregnancies. Detection of these cytokines in early pregnancies and differences in their levels may have implications for the process of successful gestation.
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Materials and methods |
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IVF
Patients were treated with standard ovulation induction protocols and underwent IVF-ET as previously described (Spandorfer et al., 1998). In brief, all women were treated with luteal phase leuprolide acetate (Lupron; Tap pharmaceuticals, Deerfield, IL), 0.51 mg s.c. daily until ovarian suppression was achieved. Ovarian stimulation was then effected with a combination of gonadotropins [HMG and/or recombinant FSH (rFSH)], employing a step-down protocol. HCG was administered (330010000 IU) when at least two follicles reached or exceeded 1617 mm mean diameter as measured by transvaginal ultrasound. Oocytes were harvested by transvaginal ultrasound-guided follicular puncture 3536 h after HCG administration.
Conventional oocyte insemination or micromanipulation was performed as indicated. The best morphologically appearing embryos were transferred into the uterine cavity 72 h after retrieval. The number of embryos transferred was dependent on maternal age, according to our standard protocol. In general, three embryos were transferred to patients under 34 years of age, patients 3439 years of age received four embryos, and patients
40 underwent transfer of up to five embryos when available. Methylprednisolone (16 mg/day) and tetracycline (250 mg every 6 h) were administered for 4 days to all patients commencing on the day of oocyte retrieval. Progesterone supplementation was initiated on the third day after HCG administration (2550 mg i.m./day) and was continued until the sonographic assessment of the pregnancy at 4751 days of gestation, as determined by the day of oocyte insemination (day 14). Sonograms were performed using transvaginal techniques.
Serum assay
All samples were centrifuged (1000 g for 15 min) and the supernatants were stored at 80°C until assayed. Serum levels of IL-2 sR, TNF-
and IFN-
were determined by means of commercial quantitative sandwich ELISAs (Quantikine, human IL-2s R
, human TNF-
and human IFN-
, Immunoassay, R&D systems, Minneapolis, MN). All cytokine serum analyses were performed simultaneously and were measured in duplicate. According to the manufacturer, the sensitivity of the assay was <10 pg/ml for IL-2 sR
, <4.4 pg/ml for TNF-
, and <8 pg/ml for IFN-
. There was no cross-reactivity with a series of cytokines and growth factors. The inter- and intra-assay coefficients of variation were 6.07.2 and 4.66.1% for IL-2 sR
, 5.47.4 and 4.25.2% for TNF-
, and 3.77.8 and 2.64.7% for IFN-
, respectively. The 96-well plates were analysed using a Multiskan Ascent kinetic analyser (Titertek Instruments Inc., Huntsville, AL) set at 450 nm. Standard curves and concentrations of the samples were calculated using Ascent V 2.4 software (Thermo Labsystems USA, Franklin, MA).
Statistical analysis
The statistical analysis was performed using StatView software (version 4.57, Abacus Concepts, Berkeley, CA). The data were analysed using analysis of variance with the Bonferroni post hoc test, the non-parametric KruskalWallis test and 2 test where appropriate. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM.
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Results |
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Serum IFN- concentrations were significantly higher (Figure 1) in patients with a biochemical pregnancy compared with patients who subsequently had a miscarriage (P = 0.008) or a normal term delivery (P = 0.005). However, serum IFN-
levels did not differ between the latter two groups (P = 0.47). Also, while serum IFN-
levels were higher in women who had an early pregnancy loss compared with women who delivered, this difference did not reach statistical significance (P = 0.14) (Figure 2).
Serum TNF- concentrations
TNF- was detectable in 83 patients (52.2%). The incidence of detectable TNF-
was higher in women with a biochemical pregnancy (60.9%) compared with patients who had a miscarriage (47.4%) or a normal term delivery (51.3%), but the difference was not statistically different. Also, there was no difference in its detection rate among patients who delivered and those who had an early pregnancy loss (51.3 versus 54.8%, respectively; P = 0.7). In general, there was no difference in pregnancy outcome based upon the ability to detect the presence of TNF-
. Further, there were no statistically significant differences in the serum concentrations of TNF-
among the three groups (Figure 1) or when comparing patients who delivered with those who had an early pregnancy loss (P = 0.4) (Figure 2).
Aetiology of infertility
In order to exclude any possible influence of the aetiology of infertility on our results, we evaluated the maternal serum levels of the examined cytokines, as well as their detection rates according to the patients aetiology of infertility, and we found no differences in their serum levels or detection rates based upon the aetiology of the patients infertility.
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Discussion |
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We found higher serum IL-2 sR levels and a higher IFN-
detection rate in women with early pregnancy loss compared with women who had a normal term delivery. Consistency in our findings was confirmed further when we evaluated cytokine serum levels and detection rates according to the final outcome. Thus, in patients with a biochemical pregnancy, serum IL-2 sR
and IFN-
levels were higher than in women who had a first-trimester miscarriage or a normal term delivery, suggesting a possible toxic effect of these cytokines at the earliest stages of human pregnancy.
These differences in the maternal serum cytokine profile, as early as 11 days after embryo transfer, according to the outcome of pregnancy are in keeping with the results of studies showing that Th-1 cytokines may play a detrimental role in embryo implantation and development. Cytokine analyses performed after the activation of peripheral blood mononuclear cells (PBMCs) with trophoblast antigens demonstrated a Th-1-type profile in women with unexplained recurrent spontaneous abortion (Hill et al., 1995). Marzi et al. (1996
) reported elevated concentrations of IL-2 and IFN-
in human spontaneous abortions compared with normal pregnancies upon antigen- and mitogen-induced activation of maternal PBMCs.
Recently, a greater Th-1 bias, with increased production of IFN- and IL-2 following stimulation of maternal PBMCs with autologous placental cells, was demonstrated in women with recurrent spontaneous abortion (Raghupathy et al., 1999
; Raghupathy, 2001
). Also, Ho et al. (2001
), who evaluated the distribution of Th-1 and Th-2 cell populations in human peripheral and decidual T cells from normal and anembryonic pregnancies, reported that the Th-1/Th-2 ratios were increased during anembryonic pregnancies. Other studies, however, suggested that the Th1/Th2 paradigm may be considered an oversimplification and that the maternalfetal relationship is not simply maternal tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions governing selective immune regulation and also control of the adhesion and vascularization processes during this dialogue (Chaouat et al., 2003
).
Recent studies have focused on measuring maternal serum levels of Th-1- and Th-2-type cytokines as a means to support previous observations regarding the Th-1/Th-2 paradigm drawn from experiments on the cytokine secretion profiles of PBMCs and decidual lymphocytes. The rationale in evaluating serum levels is based on the fact that while cytokines primarily have paracrine and autocrine effects, they often act at distant sites in the body to effect their action, and thus in certain situations they are detectable in the serum, with their levels correlating with pathological conditions (Makhseed et al., 2000).
Normal resting T and B cells do not display significant numbers of IL-2 receptors on their cell surfaces. One of the first events in the activation and release of T lymphocytes is the expression of IL-2 receptors on the plasma membranes of the cells, that are subsequently released in a soluble form into the surrounding fluid, and the secretion of a lymphokine, IL-2, that exerts its biological effects by interacting with the receptors (Rubin et al., 1985). IL-2, like other Th-1 cytokines, has been reported to have an abortogenic effect (Tezabwala et al., 1989
). However, in patients with recurrent abortions, as well as in normal pregnancies even later in pregnancy, IL-2 levels were undetectable (Makhseed et al., 2000
). Soluble IL-2 receptor has been shown to be a sensitive and quantitative marker of T-cell activation and proliferation, with activation of T cells being associated with its increased expression (Matthiesen et al., 1996
; Morris and Waldmann, 2000
). Therefore, it has been proposed that a rise in IL-2 receptor levels may indicate activation of the immune system, and thus monitoring of IL-2 receptor levels may lead to a more accurate prediction of the outcome of the pregnancy (MacLean et al., 1991
).
Matalliotakis et al. (1998) reported an increase in IL-2 receptor levels in patients with a threatened abortion who eventually had a poor outcome compared with patients with normal pregnancy. In another study, while serum IL-2 receptor levels were significantly elevated in patients with missed abortion and blighted ovum compared with patients who had normal term delivery, there was no significant difference in the IL-2 receptor levels in patients with threatened abortion compared with normal pregnant controls, leading to the conclusion that successful pregnancy is associated with significantly lower concentrations of IL-2 receptor (Gucer et al., 2001
).
MacLean et al. (2002) showed that while IL-2 receptor levels were initially significantly higher in patients with a history of recurrent miscarriage compared with non-pregnant and healthy pregnant first-trimester controls, they fell to normal in patients who eventually had a successful term pregnancy, providing a possible prognostic significance to IL-2 receptor levels at the beginning of pregnancy. Thus, a cut-off level of 1000 pg/ml, which represents 1 SD above the mean, was used to evaluate IL-2 sR
. This decision was based on the reports of other investigators proposing this level as adequate in evaluating the predictive value of IL-2 sR
on pregnancy outcome (Matalliotakis et al., 1998
). We have found that a serum IL-2 sR
concentration at 1000 pg/ml or more predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sR
1000 pg/ml versus IL-2 sR
<1000 pg/ml; P = 0.02), thus confirming the results of a previous study which reported that very high IL-2 receptor concentrations (>1300 U/ml) in patients with threatened abortion were suggestive of poor outcome (Matalliotakis et al., 1998
).
IFN- has been shown to inhibit embryonic and fetal development, as well as the proliferation of human trophoblast lines in vitro (Hill et al., 1987
, 1992; Haimovici et al., 1991
). Also, murine studies have shown a cause and effect relationship between injection of IFN-
in pregnant mice and termination of normal pregnancies (Chaouat et al., 1990
). Consistent with these, Jenkins et al. (2000
) reported higher IFN-
levels in women with miscarriage compared with the healthy pregnant group. While in another study there was no correlation between the concentrations and detection rate of IFN-
and recurrent spontaneous abortions (Makhseed et al., 2000
), in our study IFN-
-positive patients were found to have twice the risk for poor IVF pregnancy outcome compared with IFN-
-negative subjects (40.8 versus 20.0%, respectively; P < 0.02). Importantly, the detection of IFN-
influenced the implantation rate, as this was significantly lower in IFN-
-positive compared with IFN-
-negative subjects (P = 0.02), an effect which may be attributed to the cytotoxic action of IFN-
on trophoblast cells, which may inhibit implantation (Yui et al., 1994
).
TNF- has been proposed to act as a mediator of detrimental stimuli inducing embryonic death by activating apoptosis of trophoblast cells, whereas IFN-
augments TNF-mediated death of trophoblasts (Robaye et al., 1991
; Yui et al., 1994
). Injection of TNF-
to normal pregnant mice has been shown to cause abortion, whereas administration of anti-TNF-
antibodies reduced resorption rates (Gendron et al., 1990
; Arck et al., 1997
). Also, Clark et al. (1998
) reported that TNF-
together with IFN-
causes the thrombotic and inflammatory processes in maternal uteroplacental blood vessels resulting in abortion in mice (Clark et al., 1998
). Several studies have shown elevated serum TNF-
levels in patients with pathological pregnancies compared with normal pregnancies (Vassiliadis et al., 1998
; Gucer et al., 2001
).
Other studies, however, including ours have found either no difference in the detection rate and serum concentrations of TNF- among pathological and normal pregnancies, or even lower levels in the former group (Voigt and Steib, 1989
; Matalliotakis et al., 1999
). While this discrepancy may be explained by variability in the assay methods and patient population included in the various studies that may inadvertently affect the outcome, in our study the lack of difference in TNF-
levels may be as a result of the inhibiting action of the high estradiol levels in TNF-
production. Estrogen has been shown to downregulate TNF-
production and to reduce the autoimmune response in cytokine knockout mice (Deshpande et al., 1997
; Ito et al., 2001
). It may thus be proposed that during IVF stimulation cycles, where high serum estradiol levels are achieved, this inhibitory effect of estradiol may be augmentated, leading to lower TNF-
production and activity. Whether this effect also occurs locally at the feto-maternal unit, is unknown. Additionally, recent studies have proposed a possible protective role of TNF-
at the feto-placental unit with an implication in mechanisms preventing the occurrence of malformed offspring (Toder et al., 2003
). These recent reports, and our findings, are in keeping with the concept that TNF itself is not harmful in early pregnancy, as there does not seem to be correlation between TNF-
levels and early pregnancy loss (Torchinsky et al., 2003
).
This study, like previous studies that examined the possible negative effects of IL-2 sR, TNF-
and IFN-
on human pregnancy, does not address a direct cause and effect relationship between maternal Th-1 type reactivity and early pregnancy loss (Makhseed et al., 2001
; Raghupathy, 2001
). Thus, it cannot be stated conclusively that these cytokines are the cause or a result of an abnormal implantation and placentation that eventually leads to pregnancy failure.
In conclusion, our results further strengthen the currently existing notion that suggests that unexplained early human pregnancy failure might be associated with an increase in Th-1 cytokines. Furthermore, IL-2 sR and IFN-
were found to have a high predictive value in the outcome of IVF pregnancies as early as 11 days after embryo transfer. The biological significance of this finding, as well as the extent to which it can be applied clinically, remain to be evaluated by larger prospective studies.
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Submitted on November 28, 2003; accepted on January 8, 2004.
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