1 Department of Immunology, 2 Department of Obstetrics and Gynaecology, University of Liverpool, L69 3BX, UK and 3 Department of Obstetrics and Gynaecology, Niigata University School of Medicine, Japan
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Abstract |
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Key words: cytokines/peripheral blood monocytes/pregnancy outcome/recurrent pregnancy loss
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Introduction |
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Successful pregnancy may depend, at least in part, on the bias of the maternal immune response shifting away from Th1 type responses towards a Th2 phenotype, both in murine models and humans. An abnormal Th1-type cellular immune response is the basis for a recent hypothesis for immunological reproductive failure in women (Hill et al., 1995). Peripheral blood mononuclear cells (PBMC) from women with recurrent pregnancy loss (RPL) respond to trophoblast extracts in vitro by: (i) releasing soluble factors that adversely affect embryo and trophoblast viability; (ii) releasing Th1-type cytokines (TNF-
and IFN-
) and (iii) a reduction in Th2-type cytokine production (IL-4 and IL-10) (Hill et al., 1992
; Ecker et al., 1993
; Yamada et al., 1994
; Hill et al., 1995
; Raghupathy et al., 1999
). In contrast, PBMC from women with a history of successful pregnancies produce Th2-type cytokines when exposed to trophoblast extracts (Hill et al., 1995
; Raghupathy et al., 1999
). Circulating levels of TNF-
and IFN-
are higher in patients with a subsequent miscarriage compared with those with a successful pregnancy, suggesting that these cytokines may also be a potentially relevant factor in RPL patients (Müller-Eckhardt et al., 1994
; Jenkins et al., 2000
). In mouse models of RPL, the presence of Th2-type cytokines such as IL-4, IL-5, IL-6 and IL-10 appears to be associated with successful pregnancy, whilst Th1-type cytokines such as IFN-
and IL-2 are associated with embryo demise (Chaouat et al., 1990
).
Marzi et al. examined cytokine production by PBMC obtained from women throughout pregnancy (Marzi et al., 1996). Normal pregnancy was accompanied by a decrease in Th1 (IL-2 and IFN-
) productive capacity together with an increase in Th2 (IL-4 and IL-10) production, most notably in the third trimester. In five women who subsequently miscarried (a single miscarriage and not RPL), there was an increased production of Th1 cytokines (IL-2) whereas Th2 cytokines (IL-10) were reduced. A study of five women who delivered small-for-gestational-age babies demonstrated a reduced capacity for IL-10 production by PBMC, in contrast to a marked increase observed in amniotic fluid IL-10 in intrauterine growth retardation (IUGR) (Heyborne et al., 1995
), highlighting the importance of distinguishing systemic effects of pregnancy from the local effect of cytokines within the feto-placental unit.
The causes of RPL (three or more consecutive spontaneous miscarriages) are unexplained in the majority of women (Quenby and Farquharson, 1993) and it is thought that abnormalities in the immune system may have a role in idiopathic RPL (Lim et al., 1996
). Increased production by PBMC of Th1-type cytokines and decreased levels of Th2-type cytokines have been demonstrated in non-pregnant women with RPL (Hill et al., 1995
) and in RPL women at the time of miscarriage (Raghupathy et al., 1999
, 2000
).
If the shift from Th1 to Th2 cytokine response is a characteristic of normal pregnancy, a failure of this shift to occur may predispose to miscarriage in women suffering RPL. In this prospective study, the cytokine profile of PBMC from pregnant RPL women was measured and compared with the cytokine profile of pregnant women of matched gestational age with a normal obstetric history and non-pregnant women. The profile of RPL women who subsequently miscarried was also compared with those who subsequently had a live birth.
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Materials and methods |
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A total of 25 healthy pregnant women undergoing elective termination of pregnancy at 610 weeks gestation were recruited as gestationally age-matched controls. These women had no previous history of miscarriage. Eleven non-pregnant women also donated blood samples at random points in the menstrual cycle.
Cytokine production
A total of 10 ml blood was collected in vacutainer tubes containing preservative-free heparin (Sarstedt, Leicester, UK) and peripheral blood mononuclear cells (PBMC) isolated using Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Cells were washed twice by centrifugation in phosphate buffered saline and the number of viable PBMC determined by Trypan Blue exclusion. Cells were resuspended at a concentration of 1x106/ml in RPMI-1640 supplemented with 100IU/ml penicillin, 100µg/ml streptomycin and 2 mmol/l glutamine (Gibco Life Technologies Ltd., Paisley, UK) and 10% fetal bovine serum (Sigma, Poole, Dorset, UK). A total of 1x106 cells per well were cultured in 24-well plates (Nunc, Life Technologies, Paisley, UK) for 72 h at 37°C, 5% CO2 and 85% relative humidity in the presence or absence of 5 µg/ml phytohaemagglutinin (PHA), (Murex, Biotech, Dartford, UK). The cell culture medium was then harvested and replicates pooled, aliquoted and stored at 70°C for subsequent analysis.
Measurement of cytokines
Cytokine production was measured in duplicate, at one or more concentrations, using enzyme linked immunoabsorbant assay (ELISA). IL-4, IL-10 and IL-12 were measured using matched pairs of capture and detection antibodies obtained from R & D Systems (Abingdon, Oxford, UK). TNF- and IFN-
were measured using commercial kits according to the manufacturer's instructions (Genzyme, Cambridge, MA, USA) and developed using streptavidin-HRP (Zymed, San Francisco, USA) and tetramethyl benzidine (Sigma, Poole, UK). Absorbance was measured at wavelengths of 450 nm and 550 nm using a Titertek Multiskan Plus plate reader. A standard curve was constructed for each cytokine. The limit of detection of these assays was 15.6 pg/ml (TNF-
and IFN-
), 31.25 pg/ml (IL-4 and IL-10) and 7.8 pg/ml (IL-12). Cytokine production was calculated by determining the difference between PHA-stimulated and non- stimulated cells.
Statistical analysis
Statistics were calculated using Arcus software (Cambridge, UK). The data were found to be non-normally distributed and hence the non-parametric MannWhitney U-test was applied to compare differences between experimental groups. Two-sided t-tests with 95% confidence intervals were calculated with a level of significance defined at P < 0.05.
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Results |
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IL-10 was increased in pregnant women compared with non-pregnant controls, although this did not reach the selected criterion for significance (P = 0.0686). Levels of IL-10 were significantly increased in RPL patients compared with pregnant (P = 0.005) and non-pregnant controls (P = 0.026, Table I). Similar levels of IL-10 were found in RPL patients who subsequently miscarried, compared with RPL patients who subsequently had a live birth (Figure 1c
).
IL-4 production was also measured in sub-sets of pregnant women (n = 15) and those with RPL (n = 19). Levels of IL-4 were significantly increased in women with RPL (median 100 pg/ml, range 20337; P < 0.0001) compared with pregnant controls (no detectable IL-4 in any sample). Again, similar levels of IL-4 were observed in RPL patients who subsequently miscarried (n = 10; median 107 pg/ml, range 20337) compared to those who had a live birth (n = 9; median 100 pg/ml, range 75190).
One patient was sampled twice. The first sample was taken at eight weeks gestation when a fetal heartbeat was detected. At this time IFN- and TNF-
levels were similar to those in pregnant controls although IL-10 was slightly increased. The patient was sampled 2 weeks later when fetal death in utero was diagnosed. IFN-
was reduced by 90% and IL-10 was reduced by 50%. TNF-
levels were unchanged.
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Discussion |
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In this study TNF- was significantly decreased in normal first trimester samples compared with non-pregnant controls. However, in RPL women this decrease was accentuated and was even lower in RPL women who subsequently miscarried. TNF-
is an important immunoregulatory cytokine, which may be produced in Th1 or Th2-type responses and is known to have different effects depending on gestational age. It has been shown that TNF-
production by PBMC is suppressed at the mRNA level during early pregnancy and a significant increase does not occur until the eighth month of gestation (Tranchot-Diallo et al., 1997
). TNF-
production in late pregnancy is implicated in the induction of labour in mice (Chaout et al., 1990) and humans (Vince et al., 1992
). Placental production of soluble receptors has been shown to modulate TNF-
function in pregnancy (Austgulen et al., 1992
).
IFN- production was decreased in normal first trimester samples compared with non-pregnant controls, although this did not reach statistical significance. However, in RPL women this decrease was accentuated. No differences were observed between the level of IFN-
in those RPL patients who successfully completed their pregnancy compared with those who went on to miscarry.
Cytokine production by mitogen-stimulated PBMC from women in the first trimester of pregnancy showed an increase in IL-10 compared with non-pregnant controls. Although these differences did not reach statistical significance they are in agreement with similar documented changes (Marzi et al., 1996) which were augmented as pregnancy progressed. A systemic reduction in IL-10 at the time of implantation has been reported in mice (Delassus et al., 1994
). Both IL-10 and a second Th2 cytokine, IL-4, were significantly increased in women with RPL compared with pregnant controls although we did not detect any differences between the level of IL-10 or IL-4 in those RPL patients who successfully completed their pregnancy or who went on to miscarry.
Cytokines do not act in isolation but form a complex regulatory network in which modulatory interactions maintain homeostasis between the fetal unit and the maternal immune system. This complex interplay between maternal and fetal immune mechanisms also changes temporally as pregnancy progresses (Marzi et al., 1996; Tranchot-Diallo et al., 1997
). If this delicate balance is adversely affected, immunoregulatory mechanisms may be insufficient to restore homeostasis and this may lead to pregnancy failure. The central role of TNF-
in pregnancy may be important in determining the outcome of pregnancy in RPL women whose immunoregulatory network may be compromised before pregnancy occurs.
We found a greater Th1 to Th2 shift in pregnant women with RPL compared with pregnant controls of similar gestational age. This is at variance with other published studies (Makhseed et al., 1999; Raghupathy et al., 1999
, 2000
). However, these authors had taken samples after the miscarriage had been diagnosed, when the observed cytokine shift may have been the result rather than the cause of the miscarriage. The results from one RPL patient who was sampled twice, once when the pregnancy was apparently healthy according to the ultrasound and once after it had failed, add evidence to this possibility. In this patient the first sample exhibited a cytokine profile with an exacerbated Th1Th2 shift when compared with normal pregnant women, and the second (after the fetus had died) an apparent 5090% change in the Th2/Th1 balance. Other authors that found a lack of cytokine shift had sampled women having a spontaneous miscarriage rather than RPL (Marzi et al., 1996
). The pattern of cytokine production varies throughout pregnancy (Marzi et al., 1996
; Tranchot-Diallo et al., 1997
) and this should be taken into consideration when comparing data from other studies where the control group was in labour at term (Makhseed et al., 1999
).
In this study we have examined the peripheral, systemic immune response in early pregnancy. A similar and equally complex cytokine balance occurs within the endometrium and decidua (Lim et al., 1996). The degree of interaction between the two systems is yet to be determined. Peri-implantation endometrium was found to have a predominance of Th2 cytokines (Krasnow et al., 1996
; Lim et al., 1998
), and in pregnancy a 10-fold increase in decidual Th2 cytokine secretion occurred (Krasnow et al., 1996
). Women with RPL were found to have a Th1 cytokine profile in peri-implantation endometrium however, this profile did not predict pregnancy outcome (Lim et al., 2000
). In RPL women who had miscarried, more TNF-
was found in the decidua (Vives et al., 1999
) but less in the trophoblast (Lea et al., 1997
).
Cytokine production by T-cell clones derived from both the decidua and blood of women with RPL has been compared with that from non-pregnant women and those undergoing elective termination of pregnancy (Piccini and Romagnani, 1996). Although the majority of clones from all three patient groups showed a Th0-like profile (i.e. a pattern of cytokine production common to both Th1 and Th2 cells), a significantly higher number of Th1-type clones was generated from the decidua of women with RPL. In contrast, there were no differences in the cytokine profile of clones generated from peripheral blood. There was also decreased production of leukaemia inhibitory factor, IL-4 and IL-10 by decidual T cells in women with RPL (Piccinni et al., 1998). Maternal hormones may also be important regulators of cytokine production during pregnancy. Progesterone favours development of T cells producing Th2-type cytokines and even induces transient IL-4 production in established Th1 cells (Piccini et al., 1995). Hormone production could be responsible, at least in part, for the increased production of Th2-type cytokines implicated in maintenance of successful pregnancy.
PBMC from women with RPL produced increased levels of IFN- and reduced IL-10 and IL-4 when stimulated with trophoblast antigens (Hill et al., 1995
). These observations were made on non-pregnant RPL patients, which indicate that an altered profile of secretion may predispose to problems in early pregnancy. We have recently described differences in endometrial immune cell populations in RPL women prior to conception which are exacerbated in those women which subsequently went on to miscarry (Quenby et al., 1999
). This suggests that the immune system of RPL women may indeed be compromised before pregnancy occurs. The nature of this immune compromise is unclear. Genetic polymorphisms have been identified in genes coding for cytokine production (Wilson et al., 1992
; Perrey et al., 1999
; Pravica et al., 1999a
,b
). However, these polymorphisms have not been found to be more prevalent amongst women with RPL (Babbage et al., 2001
; Reid et al., 2001
).
Recurrent pregnancy loss is a pleiotrophic condition with several as yet unidentified causes. An aberration in cytokine production is implicated as a contributing factor, however further study is required to characterize its role more precisely.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 10, 2001; accepted on May 3, 2002.