High anti-paternal cytotoxic T-lymphocyte precursor frequencies in women with unexplained recurrent spontaneous abortions

Beatrix Kotlan1,5, Vilmos Fülöp2, Ágnes Padányi1, Iván Szigetvári2, Marienn Réti3, Éva Gyódi1, Éva Fehér4 and Gyözö Petrányi1

1 Department Immunogenetics, National Institute of Haematology and Immunology, Budapest, H-1113, 2 Department OB/GYN, Semmelweis University, Faculty of Health Sciences, Budapest, H-1135, 3 Department Haemapheresis, St Laszlo Hospital, Budapest, H-1097, and 4 Department Haemapheresis, National Blood Service, Budapest, H-1113, Hungary


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A number of cases of unexplained (idiopathic) recurrent spontaneous abortions may be attributable to immunological mechanisms. Several lines of evidence indicate that some immunocompetent effector cell populations play an important role in the pathogenesis of unexplained miscarriages. However a suitable method is lacking for defining an existing immunological background of recurrent spontaneous abortions. We tried to find a useful cellular immunological method, that is suitable for predicting the eventual immunological cause in the case of unexplained recurrent spontaneous abortions. We have examined the anti-paternal cytotoxic T-lymphocyte precursor frequencies by cell-mediated lympholysis and limiting dilution analysis in the peripheral blood of women with recurrent spontaneous abortions in order to reveal the functional role of this cell population in spontaneous abortions. An extremely high partner allo-antigen-specific cytotoxic T-lymphocyte precursor frequency was determined in the case of all those habitual aborters, where no other than an immunological cause could be responsible for the abortions. This phenomenon supports the important role of the T-lymphocytes in this disorder. We suggest that the immunological background of recurrent spontaneous miscarriages might be determined on the basis of a very high cytotoxic T-lymphocyte precursor frequency. This diagnostic test might be useful in selecting patients for immunotherapy.

Key words: cytotoxic T-lymphocyte precursors/immunological background/recurrent spontaneous abortion


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recurrent miscarriage is a frustrating problem for both the patient and the clinician. It affects a relatively high ratio of women, ~1% of those desiring pregnancy (Mishell, 1993Go). Primary habitual aborters are usually defined by at least three consecutive first trimester miscarriages by one partner with no live-born fetus. The causes of recurrent spontaneous abortions (RSA) are diverse (Gurka and Rocklin, 1987Go, Hill and Anderson, 1988Go; Stirrat, 1992Go). Half of recurrent miscarriages can be explained by genetic, hormonal, anatomical, metabolic abnormalities, infections or autoimmune mechanisms. Eighty percent of the unexplained pregnancy failures are proposed to have an immunological background. However the key immunopathological factor responsible for this phenomenon has not yet been identified (Johnson et al., 1986Go; Ramsden et al., 1990Go; Coulam et al., 1992Go; Stern and Coulam, 1993Go; Cook and Pridham, 1995Go; Hill, 1996Go).

The mechanism by which the haplo-identical fetus (a kind of natural allograft) is not rejected is extremely complex and is relatively little understood, although several components thought to be involved in the process have been studied extensively (Johnson and Vince, 1996Go). The protective immunological mechanisms possibly responsible for the maintenance of pregnancy, as well as a summary of data suggesting immunological mechanisms responsible for pregnancy loss, have been reviewed (Hill and Anderson, 1990; Hill, 1992Go; Hill et al., 1995Go). Extensive studies have been carried out on classical human leukocyte antigens (HLA) (Beer et al., 1985Go; Verp et al., 1993Go) and the non-classical HLA-G molecule (Carosella et al., 1999Go), as well as the characteristics of the complex trophoblast leukocyte cross reactive antigens/CD46/membrane co-factor protein antigen system (Thaler et al., 1989Go; Petranyi Gy et al., 1995b). It has become evident that normal pregnancy is characterized by a dominant harmless humoral immune response and a lack of strong maternal cell-mediated anti-fetal immunity (Wood, 1994Go; Voisin and Raghupathy, 1995Go, Bermas and Hill, 1997Go).

Although less is known about cell-mediated immune reactivity, several lines of evidence point to the role of some immunocompetent effector cell populations in the pathogenesis of unexplained repeated miscarriages (Lachapelle et al., 1996Go; Bermas and Hill, 1997Go; Marzusch et al., 1997Go; Tranchot-Diallot 1et al., 1997Go; Vassiliadou et al., 1999Go). An increased cell-mediated immune reactivity could reflect a rejection-like phenomenon (Johnson et al., 1986Go; Raghupathy, 1997Go). The association of natural killer cell activity in the human decidua (Lachapelle et al., 1996Go; Vassiliadou and Bulmer, 1996Go) and peripheral blood (Katano et al., 1997Go) with pregnancy outcome have been assessed.

The purpose of this study was to gain more information about the possible causes of unexplained RSA, and in particular, whether an immunological cause might be identified in certain cases by use of a suitable method. The question we have raised is whether cytotoxic T-lymphocytes (CTL) are involved in the course of unexplained recurrent pregnancy failure and whether their activity and the frequency of their precursors are informative in the phenomena. If anti-paternal cytotoxic T-lymphocytes are generated, they might have a central role in pregnancy outcome, as CTLs are primary mediators of allograft rejection.

Cell-mediated lympholysis (CML) and limiting dilution analysis (LDA) have been performed to study the anti-paternal cytotoxic T-cell response and also to estimate the frequency of CTL precursors (CTLpf) according to the established methods (Lefkovits and Waldman, 1984Go; Kabelitz et al., 1985Go). Identification of the number of cytotoxic precursor cells against the various HLA class I and minor H antigens in humans serves as a quantitative measurement of T-cell reactivity. As cell-mediated immune reactivity induced by transfusion (Vandeckerckhove et al., 1989Go) and rejection in transplantation (Kaminski et al., 1989Go) results in increased CTLp frequency, it was plausible to investigate partner specific CTLpf in patients with unexplained RSA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and controls
The study group consisted of 35 women (aged 22–38 years, mean ± SEM 30 ± 0.8) with a history of 2–7 miscarriages of unexplained etiology (mean ± SEM 3 ± 0.2) in the first trimester of pregnancy, without a prior live birth. All the patients were completely evaluated to rule out genetic, anatomic, endocrine, metabolic and infectious causes of RSA. The three out of the 35 cases where a relatively high level of anticardiolipin autoantibody was detected were taken into consideration when summarizing the data. The women's male partners were aged between 24 and 49 years and were found healthy in all respects. The cytogenetic investigation yielded normal results for each couple. The investigations were performed several months after the last abortion. For a relevant control group we selected 19 healthy women, undergoing elective terminations of apparently normal pregnancies. The elective terminations were all conducted during the first trimester. Their ages ranged from 20–41 years (mean ± SEM 31.5 ± 1.5). The investigations were performed ~2–3 months after the elected pregnancy termination. Seven other healthy fertile non-pregnant women (aged 25–42 years), having had at least one living child without any history of spontaneous habitual abortion, served as a second control group.

Determination of cytotoxic T-lymphocyte precursor frequency (CTLpf) by limiting dilution analysis (LDA)–cell-mediated cytotoxicity (CML)
Lymphocytes were isolated by Ficoll-Uromiro density gradient centrifugation from the sterile heparinised blood samples collected from patients with RSA some months after their last abortion. Lymphocytes were obtained from healthy non-pregnant women a few months after undergoing elective termination (in the first trimester) of apparently normal pregnancies and healthy multigravidae women served as controls. Peripheral blood mononuclear cells were isolated from the healthy male partners of patients and controls, as well as from third party people, were used for mixed lymphocyte reaction in the studies. A limited number of the wife's responder (R) cells (5x104 – 781 per well) were co-cultured with a constant number (5x104) of Mitomycin C (Sigma, Steinheim, Germany) treated (25 µg/ml) stimulator (S) cells from the husband and third party cells (in some cases). Twenty four replicate microcultures for each responder cell concentration were set up in 96 well round-bottomed microtitre plates. The RPMI 1640 culture medium (Gibco Brl, Paisley, UK) was supplemented with gentamycin (50 µg/ml), glutamine (2 mmol/l), sodium pyruvate (1%), 15% pooled human blood group AB serum and 10 IU/ml human recombinant interleukin-2 (rec IL-2, Boehringer Mannheim, Mannheim, Germany). The microcultures were incubated at 37°C in a humidified atmosphere of 5% CO2 thermostate, and fed with 100 µl fresh medium on days 2 and 6.

51Cr release cytotoxicity assay
On day 9 the generated effector cells were tested for cytotoxicity against Phytohaemagglutinin (PHA; Wellcome, Murex Diagnostics, Dartford, UK) blast cells of the original stimulator, in a standard 51Cr release assay. Briefly, PHA transformed original stimulator cells (5x106) were labelled with 400 µCi 51Cr (sodium chromate; Amersham, UK) for 90 min in 37°C waterbath and washed. From each microculture 100 µl of supernatant were removed, and after shaking the plates, 5000 (in 100µl medium) labelled target cells were added. After a 4 h incubation at 37°C in a CO2 incubator, 100µl of supernatant was removed from each well for gamma-ray counting. Total 51Cr release was assessed by incubating labelled blasts with a 1% solution of Triton-X 100 in distilled water, and spontaneous release by incubating the blasts in culture media and stimulator cells only.

Statistical analysis of CTLpf
It was assumed that the alloreactive CTL precursors are randomly and uniformly distributed in the series of cell dilutions, and that the number of CTLp per well had a Poisson distribution. For limiting dilution assays, microcultures were considered positive if their 51Cr release exceeded mean spontaneous release (24 wells containing irradiated stimulator cells and PHA blasts, but no responder cells) ± 3 SDS. From the ratio of negative wells at each responder cell concentration, the number of CTL precursors in the original cell population can be estimated, according to a standard method (Taswell et al., 1981Go). CTLpf with 95% confidence intervals was calculated using the jacknife version of the maximum likelihood method using computer software (Strijbosch et al., 1987Go).

According to established criteria (Sharrock et al., 1987Go) concerning the alloreactive T-cell repertoire in man, CTLpf values are considered as follows: very high (CTLpf >1:10 000), high (1:10 000–1:20 000), average (1:20 000– 1:60 000), low (1:60 000–1:100 000) and very low (CTLpf <100 000). Similar results could be achieved based on our investigations with healthy HLA mismatched responder stimulator pairs (only selected data presented).

Mixed lymphocyte culture reaction (MLC)
Responder cells of randomly selected women with RSA (n = 12 ) and controls (n = 10) were co-cultured with Mitomycin c-treated stimulator cells from their partners and HLA-DR different pooled third party cells for 5 days. MLC reactions were set up in triplicate, simultaneously with autologous microcultures. The proliferation of responder cells was measured by 3H-Thymidine uptake during DNA-synthesis, and evaluated by the stimulation index (SI). The stimulation index 1 (SI1) shows the degree of proliferation after allostimulation (RwomanxSpartner) in comparison with the autologous (RwomanxSwoman) level, whilst SI2 shows the proliferation against third party, HLA-DR different pooled stimulator cells. SI1 and SI2 are calculated according to the following formula:




HLA typing
HLA-A,B typing was performed by the standard NIH microlymphocytotoxicity method (Ray, 1979Go). HLA-DR and DQ antigens were determined on B-enriched lymphocyte suspensions separated from peripheral blood by the long incubation time cytotoxicity test (van Rood et al., 1975Go).

Erythrocyte antibody inhibition assay (EAI)
EAI was performed according to previously published methods (Padanyi et al., 1990Go), to measure the Fc{gamma}RII blocking activity in RSA patients. Briefly, a suspension containing B-cells from the male partner was incubated with inactivated and ultracentrifuged serum from the female partner. Human Rh(D)+ erythrocyte suspension (2%), coated previously with anti-D IgG was then added. The number of rosette forming cells was counted, and the percentage of EA rosette inhibition was calculated. Erythrocyte antibody rosette inhibition percentage (EAI%) was calculated by the following formula:

EAI% = 100x(1-EA% in test sera / EA% in control sera).

The CTLpf, MLC and EAI data of patient and control groups were analysed by Student t-statistics, and the significance of difference between the two groups was identified.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The frequency of CTL precursor cells in recurrent spontaneous aborters compaired with relevant controls
Healthy fertile non-pregnant women undergoing elective terminations of apparently normal pregnancies were used as controls. Out of 35 patients with between two and seven unexplained consecutive early miscarriages (Table IGo), 32 cases (91.4%) demonstrated an extremely high or high partner-alloantigen specific CTLpf. The highest CTLpf value in habitual aborters was 1:243 (Table IIGo). The mean CTLpf determined in women with RSA, where all other causes of RSA could be excluded, was very high: CTLpf mean = 1:4014 (SEM = 502, n = 32). Converted into a more simple form, where the number of CTL precursors present in 106 responder cells is given, that frequency would mean 249 CTL precursors/106 responder cells (Figure 1Go). The CTLpf in RSA women was significantly higher (P < 0.001) than frequencies found in healthy non-pregnant women undergoing elective pregnancy terminations of apparently normal pregnanies (CTLpf mean: 1:38 272, SEM = 2892, n = 19, that is 26 CTL precursors/106 responder cells) (Table IIIGo). It is important to note that in three cases out of the 35 patients some serum parameters showed abnormalities (irregular antibodies, anticardiolipin or anti-DNA antibodies). For these three patients further investigations were needed on autoimmune disease, and only intermediate CTLpf values (mean: 1:29 716, SEM 2060, n = 3) could be measured (Tables I and IIGoGo ). Focusing only on the 32 RSA patients without any anomaly, the partner specific CTL precursor frequency was found to be about ten times higher than in the relevant control group, indicating that an immunological background might play a role in the cause of RSA (Figure 1Go).


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Table I. Clinical data of patients. Mismatch in HLA ABC and DR antigens between patient and partner, age of patient, number of spontaneous abortions, results of gynecological, hormonal, medical examinations as well as autoimmune and serological screening
 

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Table II. Partner alloantigen specific CTL precursor frequency in patientswith recurrent spontaneous abortions.
 


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Figure 1. The mean values of partner specific cytotoxic T-lymphocyte precursor frequencies (CTLpf) determined in the RSA patient and control groups were converted pursuant to the following formula: number of CTL precursor cells/106 of responder cells. The mean CTL precursor number/106 responder cell in RSA patients was about 10 times higher compared with that of the healthy control group (healthy women undergoing elective terminations of apparently normal pregnancies, and investigated some months after an elected pregnancy termination), multigravidous women and randomly selected healthy normal responder stimulator pairs (R/S pairs normal). The CTLp number in RSA responder/stimulator pairs appear to be about five times higher compared with the control groups (R/S pairs/high HLA mismatch) (mm) with the highest HLA ABC antigen mismatch between the responder-stimulator pairs.

 

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Table III. Partner alloantigen specific CTL precursor frequency in non pregnant healthy fertile women some months after an elected pregnancy termination.
 
The mean CTLpf value of healthy control women undergoing elective pregnancy terminations of apparently normal pregnancies investigated some months after the pregnancy termination (mean: 1:38272, SEM = 2892, n = 19; 26 CTLp/106 responder cells) was very similar to that of healthy multigravidae women (CTLpf mean 1: 35 917, SEM = 5010, n = 7; 27 CTLprecursors/106 responder cells) (Figure 1Go). These CTLpf did not differ significantly from that of the randomly selected normal volunteer responder-stimulator pairs' mean CTLpf value (1:45 934, SEM = 5034, n = 10, 22 CTLprecursors/106 responder cells) (Figure 1Go).

Significantly higher cytotoxic effector cell function could be observed in patients with RSA, mainly in the high effector/target ratio range (20–70%) (individual data not shown). The autologous control microcultures showed no notable non-specific cytotoxicity, results ranged between 0–5%.

In our previous studies investigating HLA-typed, non-pregnant, randomly selected normal volunteers, we had confirmed the finding that the degree of HLA class I mismatch between responder and stimulator pairs considerably influences CTLpf. In the case of these control group members, responder-stimulator pairs with the highest HLA-antigen mismatch (4_HLA_A,B_2_HLA_DR) gave CTLpf values with a mean of 1:18 453 (54 CTLprecursors/106 responder cells). In RSA patients, about four to five times higher partner specific CTLpf could be measured, compared with this highly mismatched responder-stimulator control group. These data indicate that CTLpf values in the recurrent spontaneous aborter group are extremely high. This finding cannot be explained only by an existing HLA mismatch.

Our preliminary data with third party cells acting as stimulators show high CTLpf in five out of seven (71%) cases of RSA (mean third party CTLpf = 1:12 908, SEM = 1900, n = 7). In four out of the five cases, the CTLpf value is in the very high category (mean: 1:5209, SEM = 1828, n = 5). Further detailed investigations are needed to determine the specificity of these CTL precursors and the importance of the phenomena.

Alloantigen response in normal pregnancy and recurrent abortion
When mixed lymphocyte culture reactions were performed with responder cells of women with RSA, and stimulator cells of their partners, a high alloantigen-specific response against the partner could be measured. Partner alloantigen-induced proliferation in cultures from habitual aborter patient cells were highly positive in all but one case (SI1RSA mean: 17.75, SEM: 3.0, n = 12). Significantly (P < 0.001) higher SI indexes were observed in the RSA group than in healthy non-pregnant women undergoing elective terminations of apparently normal pregnancies (SI control mean: 3.2, SEM: 1.3, n = 10 ) (Table IVGo). A significantly (P < 0.004) higher SI was observed in patients with RSA (SI1RSA mean: 33.5, SEM 7.7, n = 12), when tested against third party pooled lymphocytes versus controls (SI control mean: 6.3 SEM 1.3, n = 10).


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Table IV. Mixed lymphocyte culture reaction obtained in the case of patients with recurrent spontaneous abortions and in healthy fertile women after an elected pregnancy termination
 
Blocking antibodies in the sera of recurrent aborters and healthy women with normal pregnancies
Sera from recurrent spontaneous aborters displayed a significantly lower rate (P < 0.05) of inhibition on erythrocyte antibody (EA) rosette formation (EAI), than those of healthy women with normal pregnancies in their case history (Figure 2Go). The mean EAI for the RSA patient group was 15.9% (SEM 2.8, n = 25) and in 20 out of 25 cases (80%) the EAI values were under 25%, which is considered to be the limit for FcR blocking antibody detection. In the sera of the healthy control women' s group, a relatively high FcR blocking activity could be found, being represented by a high EAI percentage (mean: 49.5%, SEM 5.1, n = 10).



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Figure 2. The level of blocking antibody in patients with recurrent spontaneous abortions and in healthy women with normal pregnancies in their case history was measured by an erythrocyte antibody inhibition (EAI) assay and calculated as an inhibition percentage of erythrocyte-antibody rosette formation. The mean EAI percentage in RSA patients was 15.9% compared with 49.5% in healthy pregnancies.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our data revealing extremely high partner-specific cytotoxic T-lymphocyte precursor frequencies in peripheral blood of women with RSA might have considerable importance. As this phenomena could be observed in ~97% of those RSA cases (31/32) where no other detectable cause of pregnancy loss could be found, the high CTLpf value seems to be a good indicator of a clear immunological background. The ten-fold increase in frequencies of partner-specific cytotoxic T-cell precursors, simultaneously with very high cytotoxic T-cell activities in comparison to the values in our control groups (healthy women undergoing elective terminations of apparently normal pregnancies) corroborates the importance of these cells in the induction of recurrent pregnancy failure. Our findings strengthen the investigations of Manyonda et al. (1993a), where high CTLpf could be detected in a low number of RSA cases (n = 10), while their study was focused on primigravid and multiparous women during the three trimesters of pregnancy (Manyonda et al., 1993aGo).

Our data indicate that CTLpf values in the recurrent spontaneous aborter group with nothing other than immunological cause of pregnancy loss are extremely high, and cannot be explained only by an existing HLA mismatch.

No information is available on the precursor frequency in recurrent miscarriage patients before the first conception, and it was not determined conclusively whether the CTL activity predates the first miscarriage. Furthermore, this study does not rule out the possibility that the CTLpf activity against third party cells also occurs. In most of the investigated cases we found a high or very high CTLpf against third party cells in RSA patients. Our preliminary data concerning CTL precursor frequencies against third party cells in the case of women with recurrent spontaneous abortions might suggest that the high CTLpf is a main characteristic for unexplained RSA with an immunological background, and is probably not partner specific, a possible sensitization to paternally derived antigens in women with unexplained RSA cannot be stated. In order to understand the results, it is important to take into consideration that CTLp frequency of healthy individuals against a panel of HLA class I mismatching antigens was found to be rather low (Breur-Vriesendorp et al., 1991Go). A wide range of CTLpf against different targets (cord blood, paternal and third party lymphocytes) has been found both in primigravid and multiparous women, reflecting the wide range of frequencies found in random populations (Manyonda, 1993bGo).

The high rate of paternal leukocyte-induced proliferation of recurrent spontaneous aborters' lymphocytes, found in mixed lymphocyte cultures, begs comparison with the findings of Bermas and Hill (1997) where proliferative responses to recall and alloantigens were tested in non-pregnant women with a history of three or more spontaneous abortions against relevant controls (Bermas and Hill, 1997Go). Our results show a low level of blocking antibodies in cases of women with RSA, which is in line with the hypothesis that deficiency in maternal blocking factors play a role in pregnancy loss (Rocklin et al., 1982Go; Petranyi Gy et al., 1995a; Malinowski et al., 1997Go).

Exposing the possible mechanisms that induce recurrent miscarriages might be helpful in the selection of suitable therapies. Women with a lack of allo-immune recognition might be immunised with paternal lymphocytes or thrombocytes, or receive i.v. immunoglobulin (IVIG) treatment (Beer et al., 1985Go). Detailed studies have revealed differences in some humoral immune response parameters, like the incidence of antinuclear and anticardiolipin antibodies, absence of serum factors suppressing lymphocyte proliferation in response to phytohaemagglutinin (PHA) and paternal alloantigens of non-pregnant women with unexplained RSA (Malinowski et al., 1997Go). For women with autoimmune serological abnormalities, other treatments, like pre-conception administration of aspirin, heparin and/or prednisolone are chosen (Coulam, 1995Go).

On the basis of our investigations we state that the estimation of anti-paternal cytotoxic T-lymphocyte precursor frequency seems to be a helpful and reliable clinical test for the indication of an immunological background in unexplained RSA. This finding might be of great importance in the selection of patients for immunotherapy (with paternal or third party leukocytes or thrombocytes, or syncytiotrophoblast plasma membrane preparations, or administration of IVIG) in order to facilitate a successful pregnancy outcome. Immunotherapy might be an effective treatment in affected couples, (Stephenson et al., 1998Go; Sticker et al., 2000Go) especially in those cases where the immunological cause of RSA could be proven. Until now there has remained a lack of agreement on suitable diagnostic tests defining immunological cause in unexplained RSA, although there have been some proposals on this topic involving measurement of NK cell activity (Aoki et al., 1995Go) and proliferation to recall antigens (Bermas and Hill, 1997Go).

Although a great variety of hypotheses have been proposed to explain how the haplo-identical fetus escapes immunological rejection (Gurka and Rocklin, 1987Go; Hill and Anderson, 1988Go; Hill, 1990Go; Aksel, 1992Go), and how some hormonal and biochemical factors and other immuno-regulatory mechanisms (Szekeres-Bartho et al., 1990Go) might be involved in the maintenance of pregnancy, the mechanisms leading to recurrent pregnancy loss have not yet been described. Recent studies with decidual lymphocytes (Marzusch et al., 1997Go), regulation of antigen expression (Kodama et al., 1998Go; Komatsu et al., 1998Go; Vassiliadou et al., 1999Go), and regulation of cytokine expression (Lea et al., 1995Go; Tranchot Diallot et al., 1997Go) may contribute to understand pregnancy maintenance and success. Women with unexplained RSA manifest an abnormal T-helper 1-type response following exposure to trophoblast extracts (Wegman et al., 1993Go; Hill et al., 1995Go).

We have concluded that RSA with an immunological background corresponds with extremely high CTL precursor frequencies, which might be of more than a passing interest. Further experiments are necessary to substantiate the present findings in order to clarify their role in the mechanism of RSA, such as further characterisation of these precursor cells and definition of their specificity.

On the basis of this very high rate of correlation between high CTLpf values and immunological cause of RSA, we suggest that CTLpf assay might be a useful diagnostic test for selecting RSA patients with an immunological background, for potential immunotherapy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work enjoyed the generous support of the Hungarian Ministry of Welfare (ETT 172/2000, ETT-T05 219/96). We thank Deborah J.Anderson and Joseph A.Hill from Fearing Research Laboratory, Harvard Medical School, Boston (USA), Pavol Ivanyi and Birgitta Breur-Vriesendorp from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands), and Julia Szekeres-Bartho from the Institute of Microbiology and Immunity, Semmelweis University, Pecs (Hungary) for their revisional work and suggestions.


    Notes
 
5 To whom correspondence should be addressed. E-mail: b.kotlan{at}ohvi.hu Back


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
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Submitted on September 13, 2000; accepted on February 23, 2001.





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