1 Departments of Pathology and 2 Immunology, University of Newcastle, Newcastle upon Tyne, UK
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Abstract |
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Key words: human decidua/immunosuppression/normal early pregnancy/spontaneous abortion
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Introduction |
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Molecules secreted locally in endometrium have been considered to have immunomodulatory effects on lymphocyte proliferation (Bolton et al., 1987; Parhar et al., 1988
; Pockley et al., 1988
; Matsui et al., 1989
) and natural killer cell activity (Okamoto et al., 1991
), but it is as yet unclear whether the potential of these factors to modulate immune responses differs between normal human pregnancy and spontaneous abortion. The present study aimed to address whether the profile of the molecules secreted within decidua was altered in women experiencing spontaneous early pregnancy loss compared with apparently normal fertile women, by investigating the immunomodulatory properties of unfractionated decidual cell culture supernatants on peripheral blood T-cell proliferative responses.
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Materials and methods |
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All patients had given their informed consent, and ethical committee approval had been obtained. All women undergoing elective terminations gave no history of any previous spontaneous losses or ectopic gestation.
Histological examination indicated that all samples originated from decidua parietalis. Samples showing any evidence of necrosis or acute inflammation were excluded; therapeutic and spontaneous abortion samples were morphologically indistinguishable. Apart from the exclusion criteria and consideration of gestational age, patient and control groups were randomly selected.
Decidual cell suspensions
Decidual tissue was finely minced and digested for 2x30 min at room temperature in 1 mg/ml type II collagenase and 0.1 mg/ml DNase type IV in RPMI 1640 containing 1000 U/ml penicillin, 1 mg/ml streptomycin and 2 mmol/l L-glutamine (Sigma Chemical Co., Poole, Dorset, UK). The resulting cell suspension was incubated for 10 min with 0.84% (w/v) ammonium chloride in distilled water, pH 7.2, to lyse erythrocytes, washed with RPMI 1640, resuspended in culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 1000 U/ml penicillin, 1 mg/ml streptomycin and 2 mmol/l L-glutamine), adjusted to 1x106 cells/ml and cultured for 24 h and 72 h in 25 cm3 culture flasks (Falcon cell culture products, Newcastle upon Tyne, UK). Cell-free supernatants were filtered through 0.2 µm syringe filters (Gelman Sciences, Northampton, UK) and stored at 70°C. Cell viability was assessed before supernatant collection. Culture medium in the absence of any decidual cell suspension was also incubated for the same period as test samples; `background suppression' obtained with medium incubated alone was subtracted from the immunosuppression obtained for each sample.
Effect of decidual cell supernatants on phytohaemagglutinin (PHA)-induced proliferation of peripheral blood lymphocytes
Heparinized venous blood was obtained from healthy fertile female volunteers, aged 2338 years. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation over Lymphoprep (Nycomed, Oslo, Norway) using standard techniques. They were recovered from the interface, washed twice in 0.9% saline, and resuspended in culture medium.
Decidual supernatants were added to 96-well plates in triplicate and doubling dilutions were performed in culture medium to give a final supernatant concentration ranging from 50% (v/v) to 6.25% (v/v). PHA, at a final concentration of 1.5 µg/ml and PBMC, resuspended at a concentration of 1x106 cells/ml complete medium, were added and the volume was made up to 200 µl with complete medium. Positive controls, containing cells and mitogen without any supernatant, were also set up. Cells in medium alone, without mitogen or supernatant served as negative controls. The plates were incubated for 72 h (37°C, 5% CO2 in air). At 5 h before the termination of the assay, cultures were pulsed with [3H]thymidine (Amersham Life Science, Little Chalfont, Bucks, UK) and subsequently harvested and counted in a beta-counter. The results were expressed as mean counts per minute (c.p.m.). The percentage of suppression was calculated using the following equation:
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Cell viability was checked at the end of each assay, in order to exclude the possibility that suppression was due to increased cell death with resultant cell disintegration and reduced cell numbers. For each case supernatants for both culture periods were assayed at the same time, and samples from both subject groups were assayed in each run.
Statistical analysis
Statistical analysis was performed using the MannWhitney test in order to examine whether there were significant differences in percentage suppression between the normal and the pathological groups and between the culture periods within each group. In order to check whether there was a significant association between normal pregnancy and suppression and between spontaneous abortion and enhancement, the data (mean % suppression) were inserted as a 2x2 contingency table and analysed using Fisher's exact test. Two-sided P values were calculated. The conventional significance level of P < 0.05 was adopted.
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Results |
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No significant differences in the suppressive activity of supernatants were detected between the different culture periods for both the control and the pathological groups (Table I). Results from 24 h decidual cell supernatants at 50% concentration indicated that the majority of decidual supernatants inhibited PHA-induced lymphocyte proliferation; 20 of 22 (91%) apparently normal pregnancy cases and 16 of 25 (64%) spontaneous abortion samples were inhibitory. The group of spontaneous aborters which suppressed lymphoproliferation did so at significantly increased levels compared with the therapeutic abortion samples (P = 0.04; Table I
). The remaining nine of 25 spontaneous abortion cases enhanced (28%) or had no effect (8%) on lymphocyte proliferative responses (Figure 1
). Enhancement was not observed in the therapeutic abortion samples; only two of 22 cases (9%) had no effect on lymphocyte proliferation in response to PHA (Figure 1
). At this time point and supernatant concentration, the association between stimulation of cell proliferation and spontaneous abortion, as well as suppression of cell proliferation and normal pregnancy was significant (P = 0.02).
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Samples that enhanced lymphoproliferation at 72 h also enhanced proliferation at 24 h. For those samples with immunosuppressive activity, immunoregulation was dependent on the concentration of the supernatant for all culture periods, being reduced at lower supernatant concentrations (Figure 2).
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Discussion |
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Twenty-four-hour cell suspension culture supernatants from a subgroup of spontaneous abortion cases enhanced, rather than suppressed, mitogen-induced peripheral blood lymphocyte proliferation, unlike the vast majority of 24 h therapeutic abortion cell suspension supernatants and the remaining spontaneous abortion samples, which consistently suppressed lymphoproliferation. In this regard, recent reports of significantly decreased concentrations of placental protein (PP) 14 in endometrial flushings of recurrent aborters compared with normal fertile women are of particular interest (Dalton et al., 1998). PP14 has been shown to inhibit various immunological reactions in vitro, and its immunosuppressive actions may be instrumental during implantation and subsequent pregnancy maintenance (Dalton et al., 1998
).
Separation of sporadic spontaneous abortion cases into two subgroups has been identified in previous studies (Vassiliadou and Bulmer, 1996, 1998
; Vassiliadou et al., 1999
). One subgroup possessed the same properties as the normal pregnancy control group, whereas the other group showed increased numbers of `classical' natural killer cells (Vassiliadou and Bulmer, 1996
), increased numbers of activated lymphocytes (Vassiliadou et al., 1999
) and defective cytotoxic activity (Vassiliadou and Bulmer, 1998
). The differences in immunoregulatory activity between cell culture supernatants within the spontaneous abortion group could suggest potential differences in the profile or the concentrations of cytokines and/or other soluble factors, between those spontaneous abortion patients with enhanced lymphoproliferation and those with suppressed lymphocyte proliferation responses. In the present study, longer incubation of cells from those spontaneous abortion cases with enhanced lymphoproliferation led to an increase in the immunosuppressive activity and loss of stimulatory activity, indicating that secretion by cells in decidua of soluble molecules capable of inducing suppression also occurs in vitro.
Approximately 46% of sporadic spontaneous abortions are assumed to be due to identifiable karyotypic abnormalities (Hassold et al., 1980). Since it is not possible to routinely submit all sporadic spontaneous abortion samples for karyotypic analysis in Newcastle, it is certain that a proportion of the women included in the present study experienced a spontaneous abortion due to chromosomal abnormalities. An abnormal karyotype alone, however, would not be expected to affect maternal immune function. It was postulated that those spontaneous abortion cases that are due to identifiable chromosomal abnormalities would have the same immunological response as the normal fertile women, whereas the remaining casesif there is an immunological cause for the abortionwould display an altered immunological profile. The present finding that there are two subgroups of spontaneous aborters, one displaying similar immunoregulatory activity to normal pregnancy controls, and the other showing stimulatory activity, suggests that a subgroup of women suffering spontaneous abortion exists with an altered immune profile. Undoubtedly it would be of interest to perform functional studies of sporadic abortion decidua in association with karyotypic investigation.
To date, the majority of studies aimed at elucidating the cause of early pregnancy loss, have focused on women suffering recurrent miscarriage. These patients have been considered to form a well-defined clinical subgroup in which chromosomal abnormalities are rarely the cause of miscarriage (Clifford et al., 1994). Nevertheless, recent karyotypic studies have revealed chromosomal abnormalities in approximately 55% of the aborti of primary recurrent miscarriage patients (Coulam et al., 1996
). Furthermore, heterogeneous immune responses have been seen among women suffering recurrent miscarriage (Michel et al., 1989
; Lea et al., 1995
; Bermas and Hill, 1997
), in common with the results obtained in our studies in women experiencing sporadic spontaneous pregnancy loss. Sporadic aborters include even women who will eventually be classified as recurrent aborters. Hence, if there is an immunological cause for the miscarriage in these patients, this should possibly be identified from the first or the second miscarriage, while these women are still considered sporadic aborters. Nonetheless, results from studies in spontaneous miscarriage patients should be interpreted with caution, since they could reflect the consequence rather than the cause of miscarriage. However, it is unlikely that the changes noted in the spontaneous abortion group merely reflected the non-vital state of the pregnancy, since decidual samples showing evidence of necrosis or acute inflammation were excluded from the study.
Significantly increased levels of suppression were detected from those spontaneous abortion cell culture supernatants that suppressed lymphoproliferation, when compared with therapeutic abortion samples. It is unlikely that this difference is solely due to in-vitro cell activation and hence increased in-vitro secretion of suppressor factors in spontaneous abortion samples, since the same isolation procedure was employed in apparently normal pregnancy samples. An explanation could be that considerable amounts of suppressor factors are already present in situ in decidua in this subgroup of spontaneous aborters. Significantly increased numbers of activated leukocytes have been found in decidua of women suffering spontaneous miscarriage when compared with normal pregnancy controls by flow cytometry (Maruyama et al., 1992). The results have been extended in immunohistochemical studies which have detected increased numbers of CD69-positive and CD25-positive T cells within decidua in a proportion of spontaneous abortion cases (Vassiliadou et al., 1999
); it is possible that in this subgroup of spontaneous abortion cases these cells are in an activated state in situ and hence show altered secretion of suppressor molecules compared with apparently normal pregnancy decidua.
Enhancement was not observed in therapeutic abortion cases. It is noteworthy however, that occasional samples had no effect on PHA-induced lymphoproliferation. Women undergoing elective pregnancy terminations are considered to be the best available control group. Nevertheless, it is not certain whether they can be considered as truly normal, since the outcome had there been no intervention is unknown. Hence, it is possible that these women who had no immunosuppressive properties, although they were undergoing apparently normal pregnancies, were destined for a different pregnancy outcome from the remainder.
The present study confirmed that innate immunosuppression within decidua is a prerequisite for pregnancy maintenance, since it was detected in the vast majority of normal fertile women, while a proportion of spontaneous abortion cases can yield altered immune responses which could be associated with poor pregnancy outcome. Future studies should also focus on functional assays utilizing purified decidual cell populations from apparently normal pregnancy samples, as well as fully karyotyped miscarriages, in an effort to unveil the contribution of each cell type in the regulation of immune responses at the maternalfetal interface.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 22, 1999; accepted on May 27, 1999.