Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions

M. Makhseed1,4, R. Raghupathy2, F. Azizieh2, A. Omu1, E. Al-Shamali3 and L. Ashkanani3

1 Departments of Obstetrics & Gynecology and 2 Microbiology, Faculty of Medicine, Kuwait University and 3 Maternity Hospital, Kuwait


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: This study compared Th1–Th2 cytokine profiles in a subgroup of recurrent aborters who had an abortion with those in a subgroup of recurrent aborters who had a successful pregnancy. METHODS: Fifty-four women with a history of at least three normal pregnancies, 24 women with a history of recurrent spontaneous abortion (RSA) followed by abortion (RSA->A) and 39 women with a history of RSA followed by normal pregnancy (RSA->N) were studied. Blood samples and placentas were obtained at the time of delivery or abortion; peripheral blood mononuclear cells were stimulated separately with phytohaemagglutinin and with autologous placental cells, and the secreted cytokines estimated. RESULTS: Peripheral blood mononuclear cells from the RSA->N subgroup secreted higher concentrations of Th1-type cytokines as compared with normal pregnant women, indicating a higher Th1 bias in these women. However, women in the RSA->N subgroup had significantly higher concentrations of Th2 cytokines as compared with women in the RSA->A subgroup. A comparison of Th1:Th2 cytokine ratios indicated a higher Th2 bias in RSA->N women as compared with RSA->A women. CONCLUSIONS: We conclude that abortion-prone women who proceed to have successful pregnancy are more Th2-biased than abortion-prone women who abort, and that recurrent aborters who undergo spontaneous abortion have a stronger Th1 bias than aborters who have normal pregnancy.

Key words: pregnancy/recurrent spontaneous abortion/Th1 cytokines/Th2 cytokines


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cellular immune effector mechanisms have been proposed as being responsible for at least a proportion of `unexplained' recurrent spontaneous abortions (RSA) (Hill, 1995Go; Raghupathy, 1997Go). Unexplained RSA accounts for about 40–60% of all cases of RSA (Stray-Pederson and Stray-Pederson, 1984Go). Recent evidence, much of it emerging from research on animal models, indicates important roles for T cells, macrophages, natural killer (NK) cells and cytokines in causing pregnancy failure (Wegmann et al., 1993Go; Hill, 1995Go; Chaouat and Menu, 1997Go; Raghupathy, 1997Go; Clark, 1999Go; Clark et al., 1999Go). Clark and colleagues have proposed that an abortogenic pathway consisting of macrophages, NK cells and T cells and the cytokines tumour necrosis factor (TNF), interferon (IFN)-{gamma} and interleukin (IL)-2 may underlie immunologically mediated abortions (Clark et al., 1998Go). Resorption sites in mice are infiltrated by NK cells (Gendron and Baines, 1988Go) and activated macrophages (Baines et al., 1997Go). The administration of the cytokines TNF-{alpha}, IFN-{gamma} or IL-2 to normal pregnant mice causes abortions (Chaouat et al., 1990Go; Tezabvala et al., 1989Go). TNF-{alpha} stimulates the programmed death of human primary villous trophoblast cells, and IFN-{gamma} augments TNF-mediated killing of trophoblasts (Yui et al., 1994Go).

IL-2, IFN-{gamma} and TNF-ß, i.e. cytokines that have deleterious effects on the conceptus, are cytokines that are characteristic of so-called Th1-type cells, which induce several cytotoxic and inflammatory reactions and are thus responsible for the induction of cell-mediated inflammatory reactions (Mosmann and Coffman, 1989Go; Romagnani, 1994Go; Mosmann and Sad, 1996Go). Th2 cells, on the other hand, secrete IL-4, IL-5, IL-6, IL-10 and IL-13 and are associated with help for humoral immunity. Based on the observations of deleterious effects of Th1-type cytokines on murine pregnancy and on observations that pregnancy appears to be associated with a down-regulation of cell-mediated immunity manifested as reduced delayed-type hypersensitivity (DTH) (Holland et al., 1984Go), NK activity (Luft and Remington, 1984Go) and weakened resistance to intracellular infections (Wegmann et al., 1993Go), it was suggested that pregnancy is associated with a down-regulation of Th1-type activity and enhancement of Th2-type activity (Wegmann et al., 1993Go; Hill, 1995Go; Raghupathy, 1997Go).

In humans, elevated concentrations of the Th1-type cytokines IL-2 and IFN-{gamma}, and decreased concentrations of the Th2-type cytokine IL-10, have been reported in spontaneous abortion upon antigen- and mitogen-induced activation of maternal peripheral blood mononuclear cells (PBMC) (Marzi et al., 1996Go). The activation of PBMC with trophoblast antigens followed by cytokine analyses has shown that women with unexplained RSA have a Th1-type profile (Hill et al., 1995Go). We have recently demonstrated increased production of IFN-{gamma}, TNF-{alpha}, TNF-ß and IL-2 by mitogen-activated PBMC from women with RSA and, conversely, increased production of IL-4, IL-5, IL-6 and IL-10 by PBMC from women with normal pregnancy at the end of the first trimester (Raghupathy et al., 2000Go) and at delivery (Makhseed et al., 1999Go). We have shown a similar Th1 bias in unexplained RSA and a Th2 bias in successful pregnancy by comparing cytokine production after stimulation with autologous placental cells and with trophoblast antigens (Raghupathy et al., 1999Go). A recent study showed significantly higher concentrations of IL-4- and IL-10- producing T cell clones generated from cells infiltrating the decidua of women with normal pregnancy as compared with those experiencing unexplained RSA (Piccinni et al., 1998Go). Indeed, it has been demonstrated (Ekerfelt et al., 1997Go) that there is a significantly larger number of IL-4-secreting maternal cells in response to stimulation with paternal leukocytes as compared with other, unrelated leukocytes, suggesting a selective and specific immune deviation towards Th2 bias in normal pregnancy. Taken together, these data provide significant support for the contention that women with normal pregnancy have a higher Th2 bias, whereas women with a history of unexplained RSA have a bias towards Th1-type reactivity.

This communication focuses on a subgroup of women who have a history of recurrent abortions but go on to have a successful pregnancy. We felt it to be of great interest to determine the Th1–Th2 cytokine profiles in this group of women and to compare these profiles with those in recurrent aborters who have another spontaneous abortion. The objective of this study was to compare the cytokine profiles in abortion-prone women who had a successful pregnancy (RSA->N) with abortion-prone women who had a spontaneous abortion (RSA->A) and with women who have had a history of at least three successful pregnancies and no previous abortions (N). Cultures of maternal PBMC obtained at the time of abortion or delivery were stimulated with the mitogen phytohaemagglutinin (PHA) and with autologous placental cells, after which cytokine concentrations were estimated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Subjects were selected at the Kuwait Maternity Hospital from patients with three or more previous abortions who were routinely referred to the Recurrent Abortion Clinic for investigation into possible anatomical, endocrinological, infectious, genetic and immunological causes of abortion. Immunological tests consisted of assays for anti-nuclear antibodies, lupus anticoagulant and anti-cardiolipin antibodies. The normal-control group consisted of 54 women who were in labour with a single fetus, who had previously had at least three normal pregnancies, with no history of abortion, ectopic pregnancy, pre-term delivery or stillbirth. Twenty-four of these women were also tested at the end of the first trimester as a gestational stage control for the RSA group. The RSA group consisted of a total of 63 women who had had at least three previous unexplained miscarriages and who had been investigated clinically. All couples were screened for balanced translocations and the women were examined by hysterosalpingography. These subjects had no demonstrable cause for their pregnancy losses. Of these women, one subgroup of recurrent aborters, consisting of 39 women, had their first successful pregnancy in 1997–1998; this subgroup was termed RSA->N. The remaining 24 women with a history of RSA underwent another spontaneous abortion and were termed RSA->A. Blood samples from these women were collected prior to evacuation. This study has the approval of the Ethics Committees of the Maternity Hospital and of the Faculty of Medicine, Kuwait University; informed consent was obtained before blood samples were collected.

Mitogen-induced stimulation of PBMC
Peripheral blood was obtained by venepuncture from the normal pregnant subjects at the time of delivery and from the aborters on the day of abortion. PBMC were separated by Ficoll-paque (Pharmacia Biotech, Sweden) density gradient centrifugation, suspended in Roswell Park Memorial Institute (RPMI) medium (Gibco BRL, USA) containing 10% fetal calf serum, aliquoted into 96-well tissue culture plates at a density of 105 cells per well and then challenged with PHA (Sigma Chemicals, USA) at a concentration of 5 µg/ml for a period of 96 h. Supernatants were harvested at 24 and 96 h; some of the wells were pulsed with [3H]thymidine (1 µCi per well) for assessment of mitogen-induced proliferation. Thymidine-pulsed wells were harvested 18 h later and the radioactivity estimated. The stimulation index (SI) was calculated as a ratio of thymidine uptake by PHA-stimulated cells to that by non-stimulated cells.

Mixed lymphocyte–placenta reaction (MLPR)
Placentas from normal delivery and from abortions were obtained immediately after spontaneous vaginal delivery. Single cell suspensions from placentas were prepared as described (Kauma et al., 1991Go). One or two cotyledons were removed from the underlying fibrous tissues and transferred to sterile containers containing RPMI medium (Gibco BRL) supplemented with penicillin, streptomycin, fungizone and 10% fetal bovine serum. The placental tissue was rinsed thoroughly with RPMI medium to reduce red blood cell contamination. The outermost (maternal face) tissue was removed and tissue from the interior of the placenta harvested aseptically, minced and washed thoroughly in several changes of medium. Approximately 30g of placental tissue was digested for 12 h at 37°C with 3 ml of 50 units/ml of the enzyme dispase (Collaborative Research Inc., Bidford, MA, USA) in RPMI 1640 medium containing 10% FBS and antibiotics. After digestion, the mixture was passed through gauze pads to remove undigested fragments, and then subjected to Ficoll-hypaque density gradient sedimentation. The interface consisting of mononuclear cells was harvested, washed three times in RPMI and resuspended in medium for counting and viability testing. The 12 h treatment with dispase and subsequent density gradient sedimentation routinely yielded single cell suspensions with viability ranging from 80–97%.

Maternal anti-placental cell reactivity was examined by co-culturing maternal PBMC (responders) with irradiated placental cells (stimulators) as described previously (Raghupathy et al., 1999Go). Maternal PBMC were obtained by Ficoll density gradient sedimentation and resuspended in RPMI medium at 106 cells/ml. Placental cells were irradiated in a gamma chamber at 3000 rads, washed and then resuspended at 106 cells/ml. One hundred microlitres each of the PBMC suspension and placental cell suspension were added to wells of 96-well tissue culture plates and the plates incubated for 96 h at 37°C, 5% CO2, 90% humidity. Culture supernatants were collected 24 and 96 h later. Some of the wells were pulsed with [3H]thymidine at 96 h and the uptake of thymidine measured 18 h later. Stimulation indices (SI) were calculated as the ratio of thymidine uptake by stimulated cells to that by unstimulated, control cells.

Determination of cytokine concentrations
IL-2, IL-4, IL-5, IL-6, IL-10, TNF-{alpha} and IFN-{gamma} were assayed by enzyme-linked immunosorbent assay (ELISA) using kits obtained from Immunotech SA (Marseilles, France) and TNF-ß by ELISA kits obtained from R&D Systems (Minneapolis, MN, USA). The manufacturer's protocols were followed for the assays. Standard curves were plotted for each of the cytokines using reference recombinant cytokines and the results were read from these curves. The sensitivities of each of the assays were as follows: 5 pg/ml for IL-2, 10 pg/ml for TNF-{alpha}, 7 pg/ml for TNF-ß, 3 pg/ml for IFN-{gamma}, 5 pg/ml for IL-4, 1 pg/ml for IL-5, 3 pg/ml for IL-6 and 5 pg/ml for IL-10.

Statistical analysis
The Mann–Whitney U-test was used for non-parametric comparisons of median cytokine concentrations, as the data were not normally distributed.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Proliferative responses of PBMC
Stimulation indices (SI) of the PBMC from the RSA->N group ranged from 1 to 866 with a median of 50, while the SI of the RSA->A group ranged from 1 to 600 with a median of 82. No significant differences were apparent in the median SI of the two groups.

Secretion of Th2-type and Th1-type cytokines in mitogen-stimulated cultures
Women with a history of recurrent abortions who had subsequent normal delivery (RSA->N) had Th2 cytokine concentrations similar to those in women with a history of successful pregnancy (N); of the four Th2 cytokines tested at two different time points, only IL-5 tested at 96 h was produced at significantly higher concentrations (P < 0.002) by the normal pregnancy group (N) as compared with the RSA->N group (Figures 1 and 2GoGo). However, the RSA->N subgroup, as compared with the RSA->A subgroup, produced significantly higher concentrations of IL-4 (P < 0.002) and IL-5 (P < 0.002) at the 24 h point and significantly higher concentrations of IL-6 (P < 0.002) at the 96 h point. The general trend was towards higher production of all four Th2 cytokines at both the time points in the RSA->N group as compared with the RSA->A group, even though only those mentioned above were statistically significant (Figures 1 and 2GoGo). In comparison with the RSA->A group, the normal pregnancy first trimester group produced significantly higher concentrations of IL-6 (P < 0.0001) and IL-10 (P < 0.0001) at both time points and IL-4 (P < 0.005) at the 24 h time point.



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Figure 1. Median concentrations of Th2-secreted cytokines produced by mitogen-stimulated peripheral blood mononuclear cell culturesat 24 h. In this and all subsequent figures, the graphs depict median values, 95% confidence ranges, 25th and 75th percentiles.IL = interleukin; first = first trimester, normal pregnancy; RSA-A = recurrent aborters who have had another abortion;RSA-N = recurrent aborters who have had a successful pregnancy; N = normal pregnancy at delivery.

 


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Figure 2. Median concentrations of Th2-secreted cytokines produced by mitogen-stimulated peripheral blood mononuclear cells cultures at 96 h. For abbreviations, see Figure 1Go.

 
Except for a significant decrease (P < 0.002) in the concentrations of TNF-{alpha} at 96 h, concentrations of Th1 cytokines produced by the RSA->A group were not different from those produced by the RSA->N group (Figures 3 and 4GoGo). In 24 h cultures, concentrations of Th1-type cytokines were significantly higher in the RSA->N group than in the normal pregnancy group at delivery (Figure 3Go).



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Figure 3. Median concentrations of Th1-secreted cytokines produced by mitogen-stimulated peripheral blood mononuclear cells cultures at 24 h. IFN = interferon. For other abbreviations, see Figure 1Go.

 


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Figure 4. Median concentrations of Th1-secreted cytokines produced by mitogen-stimulated peripheral blood mononuclear cell cultures at 96 h. IFN = interferon. For other abbreviations, see Figure 1Go.

 
In general, these mitogen-stimulation experiments indicate that women in the RSA->N group produce higher concentrations of Th2 cytokines than the RSA->A group, but more or less similar concentrations of Th1 cytokines. Keeping in mind the fact that the balance of Th1 and Th2 cytokines is probably of greater importance than their concentrations alone, we calculated the ratios of various Th1 to Th2 cytokines.

Ratios of Th1:Th2 cytokines in mitogen-stimulated cultures
The ratios of mean Th1 to Th2 cytokine concentrations in the RSA->A group were consistently higher than the ratios in the gestational stage control group of first trimester normal pregnancy (Table IGo). The RSA->N group showed a greater bias towards Th1 reactivity than the first trimester normal pregnancy group but substantially lower than the RSA->A group.


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Table I. Ratio of Th1- to Th2-secreted cytokines produced by mitogen-stimulated peripheral blood mononuclear cells
 
In all combinations evaluated, the RSA->A group showed higher Th1:Th2 cytokine ratios than the RSA->N group. For example, the IFN:IL-4 ratio at the 24 h point is 182 in the RSA->A group, i.e. almost 17-fold higher than the IFN:IL-4 ratio in normal pregnancy at delivery and 23-fold higher than that in normal pregnancy at the first trimester, while the ratio in the RSA->N group is 24, only about 2-fold higher than that in normal pregnancy, but still substantially lower than the ratio in RSA->A women. Similarly, the IFN:IL-10 ratio at the 24 h point is 15 times higher in RSA->A women as compared with normal pregnancy at delivery and 30 times higher as compared with normal pregnancy at the first trimester, but only 4-fold higher in RSA->N women. Thus, while the Th1:Th2 ratio is higher in abortion-prone women who had a normal pregnancy as compared with women with a history of successful pregnancy, the Th1:Th2 ratio in recurrent aborters with normal delivery is much lower than abortion-prone women who go on to have another abortion, indicating a lower Th1 bias in recurrent aborters who have successful pregnancy.

Secretion of Th1-type and Th2-type cytokines in MLPR-stimulated cultures
IL-2, IL-4 and IL-5 were not measurable in these culture supernatants. Women in the RSA->N group tended to produce higher concentrations of IL-6 and IL-10 than did women in the RSA->A group but the differences were not statistically significant (data not shown). A comparison of the ratios of mean concentrations of Th2 to Th1 cytokines provides a better picture of Th1-versus-Th2 status (Table IIGo). A greater value in this combination indicates a greater Th2 bias; we find that the Th2:Th1 ratios in RSA->N women are closer to those in normal pregnancy (N) and substantially higher than those in RSA->A women in all of the permutations. For example, the IL-10:IFN-{gamma} ratio in the RSA->N group at 24 h is 72, a value twice that in normal pregnancy but 72-fold greater than that in the RSA->A group. These data support the notion of a greater Th2 bias in recurrent aborters who had a normal pregnancy than in recurrent aborters who aborted their pregnancies.


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Table II. Ratio of Th2- to Th1-secreted cytokines produced by mixed lymphocyte-placenta reaction-stimulated peripheral blood mononuclear cells
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Spontaneous abortion is the most common form of pregnancy failure, with 20% of pregnancies culminating in detectable spontaneous abortions. The risk of a subsequent miscarriage after one spontaneous loss is about 24% and is 26% after two and 32% after three consecutive losses (Warburton and Fraser, 1961Go). Thus, women who have had three consecutive spontaneous abortions, as did the women in this study, have a 68% probability of having a successful pregnancy. The current study was prompted by an interest in elucidating the immunological milieu in which habitual aborters have a successful pregnancy and in particular to ascertain whether their cytokine profiles are more conducive to successful pregnancy as opposed to habitual aborters who continue to abort. We proposed that abortion-prone women who go on to have a successful pregnancy may be more Th2-biased (or less Th1-biased) as compared with abortion-prone women who continue to abort. To test this hypothesis, we stimulated PBMC with a mitogen and with autologous placental cells as a means to ascertain the status of Th1 versus Th2 reactivity.

The mitogen-stimulation experiments indicate a greater Th1 bias in abortion-prone women who abort as compared with abortion-prone women who have a normal pregnancy. The placental cell-stimulation experiments also point to a greater Th2 bias in abortion-prone women who proceed to have a normal pregnancy as opposed to abortion-prone women who abort. This conclusion is supported by the comparison of Th1:Th2 cytokine ratios (Tables I and IIGoGo). Relative differences in these ratios between normal pregnancy and RSA may be indicative of a difference in bias towards Th1/Th2 reactivity.

In the mitogen-stimulation experiments we were able to include normal controls of similar gestational age to the RSA samples; we measured cytokine production by samples obtained from normal first trimester pregnancies. This was done to take into consideration the possibility of differences in cytokine profiles towards the end of the first trimester and at the end of gestation. In fact, we found statistically significant differences in the concentrations of IL-6, IL-10, TNF-{alpha} and IFN-{gamma} measured at these two time points, in normal pregnancy. This serves to highlight the importance of comparing cytokine production from patients and controls from the same length of gestation. In these experiments, significant differences were observed in the concentrations of IL-4, IL-6, IL-10 and IL-2 between the RSA->A group and first trimester normal controls. We have previously demonstrated that Th1 cytokines are produced at higher concentrations by the women with a history of RSA than by women with a history of normal pregnancy in the first trimester (Raghupathy et al., 2000Go) and at delivery (Makhseed et al., 1999Go). Thus, women with a history of recurrent abortion produce higher concentrations of Th1 cytokines than normal pregnant women, whether or not the recurrent aborters had a subsequent successful outcome or yet another abortion.

It was not possible to evaluate responses of maternal lymphocytes to placental cells in normal pregnancy at the first trimester for obvious reasons. Thus, in the MLPR experiment, comparisons were only possible between abortions occurring at the end of the first trimester and normal pregnant women at delivery. Given this limitation, the results of the MLPR experiments may have to be evaluated cautiously.

It must also be pointed out that the nature of the antigenic stimuli and the stimulating cells in the MLPR experiments are unidentified. The placental cell suspensions used in this study consisted of varying numbers of trophoblast cells, leukocytes etc. and we were not able to normalize the numbers of the various cell types. This caveat, however, does not apply to our mitogen-stimulation experiments, which are expected to provide information on the overall bias of the peripheral immune system towards Th1 versus Th2 reactivity.

This study, like other studies on human pregnancy failure, does not address a direct cause-and-consequence relationship between maternal Th1-type reactivity and spontaneous abortion. For example, an inflammatory maternal response to a fetus that has died due to non-immunological factors, could still involve a Th1 profile, in which case the Th1 bias in spontaneous abortion could be secondary to fetal death. Much of the work on recurrent spontaneous abortions in humans has focused on the analyses of maternal responses and local changes that occur following abortion. To obtain evidence for a clearer association between cytokine profiles and pregnancy loss, comparative investigations of maternal cytokine profiles in successful and unsuccessful pregnancy at frequent intervals from early pregnancy onwards is needed; thus, if changes are demonstrated in cytokine profiles either in the periphery or at local uterine or placental sites, before pregnancy loss is observed, it would be possible to make a stronger link between cytokine patterns and pregnancy loss. Besides this, clearly there is also a need for a better understanding of changes in the local milieu at the maternal–fetal interface at various gestational stages, in normal pregnancy versus unexplained spontaneous abortion.

IL-10 has emerged as an important Th2-type cytokine in the maintenance of pregnancy. In animal models it has been shown that the injection of IL-10, an anti-inflammatory Th2-type cytokine with pronounced down-regulatory effects on Th1-type cytokines, prevents fetal wastage in mice prone to fetal resorption (Chaouat et al., 1995Go, 1996Go). Thus, it is conceivable that increased Th2 bias, manifested as increased production of IL-10, may well be conducive to successful pregnancy in women with immunologically mediated RPL. It is directly involved in down-regulating Th1-type activity by inhibiting the production of IFN-{gamma} (Fiorentino et al., 1989Go) which has been shown to have deleterious effects on pregnancy (Tezabvala et al., 1989Go; Chaouat et al., 1990Go; Clark et al., 1998Go). In fact, it has been proposed that an important immunoregulatory role for IL-10 is the maintenance of Th2 bias and the induction of a shift away from Th1 bias (Romagnani, 1994Go). Thus, while the increased production of Th2-type cytokines in habitual aborters who have a successful gestation may not be due to any direct effects of these cytokines on the conceptus, they may well aid the survival of the conceptus by down-regulating Th1-type reactivity, which is detrimental to the success of pregnancy.

The data described in this paper would suggest that women with a history of recurrent spontaneous abortion presumably mediated by Th1-type reactivity may be able to have a successful pregnancy if their profile shifts to Th2 dominance. While this study does not provide proof that the abortion-prone women had a successful pregnancy because of a shift towards Th2 bias, evidence provided here suggests that this might be the case.

We propose that a shift in the bias of overall maternal reactivity to the more conducive Th2 type, or at least a reduction in strong Th1 bias, may favour pregnancy and may thus provide the right milieu for a woman with immunologically mediated recurrent abortion to have successful pregnancy. If these observations are validated, it may pave the way for the development of methods to manipulate the maternal immune system towards a Th2-type situation that would favour the success of pregnancy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by a grant from the Kuwait Foundation for the Advancement of Sciences (KFAS 95-07-10).


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Faculty of Medicine, Kuwait University, P.O.Box 24923, Kuwait 13110. Back


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 Materials and methods
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 Discussion
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Submitted on March 30, 2001; accepted on July 9, 2001.