Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss?

M.D. Bates1, S. Quenby2, K. Takakuwa3, P.M. Johnson1 and G.S. Vince1,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Successful pregnancy may depend on a Th2-type cytokine response, whilst, conversely, a poor pregnancy outcome may be associated with an increase in Th1 cytokines and a concomitant decrease in Th2 cytokines. This prospective study was designed to elucidate whether a failure of the cytokine shift pre-dated miscarriage and was therefore likely to be an aetiological factor in recurrent pregnancy loss (RPL). METHODS: Cytokine production by stimulated peripheral blood mononuclear cells from 46 pregnant women who had previously suffered idiopathic RPL during early pregnancy was compared with 25 gestationally age-matched pregnant controls and 11 non-pregnant women. RESULTS: Production of IFN-{gamma} was lower in pregnant than in non-pregnant women and even lower in RPL pregnant women (P = 0.0191). IL-10 was increased in pregnant women compared with non-pregnant controls, and further increased in RPL patients (P = 0.026). IL-4 was also increased in women with RPL (P = 0.0001). No differences in IFN-{gamma}, IL-10 or IL-4 secretion were observed in RPL patients who subsequently miscarried compared with those who successfully completed the pregnancy. RPL women with a successful reproductive outcome had similar concentrations of TNF-{alpha} to pregnant women, RPL women who subsequently miscarried had significantly lower levels than either pregnant women (P = 0.02) or non-pregnant controls (P = 0.0004). CONCLUSIONS: Contrary to our hypothesis, the cytokine shift, which appears to characterize normal pregnancy, was accentuated rather than diminished in RPL pregnant women.

Key words: cytokines/peripheral blood monocytes/pregnancy outcome/recurrent pregnancy loss


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recent attention has focused on elucidating the immunobiological roles of cytokines in normal human pregnancy following the accumulated reports of complex cytokine activity within uteroplacental tissues (Robertson et al., 1994Go; for review). T helper (Th) cells can differentiate into subsets with distinctive patterns of cytokine release. It has been proposed that Th1-type responses (e.g. the production of IL-2 and IFN-{gamma}) are systemically suppressed in murine pregnancy and that local expression of Th2-type cytokines (e.g. IL-4, IL-6, IL-10) in placental tissue might be beneficial for fetal survival (Wegmann et al., 1993Go). Whether an analogous situation exists in human pregnancy is unclear as yet (Vince and Johnson, 1996Go), although partial systemic impairment of Th1 responses is compatible with clinical evidence that a number of infectious diseases caused by intracellular pathogens can sometimes be exacerbated in pregnancy, e.g. cytomegalovirus and malaria (Hart, 1988Go). Furthermore, rheumatoid arthritis, characterized by a Th1 response, often undergoes remission during pregnancy (Da Silva and Spector, 1992).

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., 1995Go). 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-{alpha} and IFN-{gamma}) and (iii) a reduction in Th2-type cytokine production (IL-4 and IL-10) (Hill et al., 1992Go; Ecker et al., 1993Go; Yamada et al., 1994Go; Hill et al., 1995Go; Raghupathy et al., 1999Go). In contrast, PBMC from women with a history of successful pregnancies produce Th2-type cytokines when exposed to trophoblast extracts (Hill et al., 1995Go; Raghupathy et al., 1999Go). Circulating levels of TNF-{alpha} and IFN- {gamma} 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., 1994Go; Jenkins et al., 2000Go). 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- {gamma} and IL-2 are associated with embryo demise (Chaouat et al., 1990Go).

Marzi et al. examined cytokine production by PBMC obtained from women throughout pregnancy (Marzi et al., 1996Go). Normal pregnancy was accompanied by a decrease in Th1 (IL-2 and IFN- {gamma}) 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., 1995Go), 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, 1993Go) and it is thought that abnormalities in the immune system may have a role in idiopathic RPL (Lim et al., 1996Go). 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., 1995Go) and in RPL women at the time of miscarriage (Raghupathy et al., 1999Go, 2000Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and controls
Local ethical committee approval was obtained for this study. Venous blood samples (10 ml) were obtained from 46 pregnant women attending the Recurrent Miscarriage Clinic at the Liverpool Women's Hospital having previously suffered at least three consecutive miscarriages. Patients were asked to phone the clinic as soon as they realised that they were pregnant and were seen as soon as possible thereafter. They were recruited into the study if an ultrasound scan showed an amniotic sac containing a yolk sac or an intrauterine fetus between 6–10 weeks gestation. Patients were seen fortnightly with ultrasonography for reassurance until 12 weeks gestation and subsequently received routine antenatal care. Patients were excluded from the study if one of the following causes for their miscarriages was found; antiphospholipid syndrome, endocrine cause (oligomenorrhoea, abnormal thyroid function tests), chromosomal cause (maternal or paternal balanced translocation) or uterine structural abnormality (assessed by cervical weakness only) (Drakeley et al., 1998Go). One patient was sampled at eight weeks gestation when a fetal heartbeat was detected and two weeks later when death in utero was diagnosed.

A total of 25 healthy pregnant women undergoing elective termination of pregnancy at 6–10 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-{alpha} and IFN- {gamma} 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-{alpha} and IFN- {gamma}), 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 Mann–Whitney 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the 46 pregnant recurrent miscarriage patients recruited in this study, 33 subsequently produced a live birth and 13 miscarried. The production of cytokines by in-vitro stimulated PBMC was measured in blood samples obtained from pregnant women with RPL and compared with normal pregnant women of matched gestational age and non-pregnant controls (Table IGo). Basal levels of cytokine production by unstimulated cultured PBMCs were significantly increased upon stimulation with PHA.


View this table:
[in this window]
[in a new window]
 
Table I. Median cytokine production by PBMC from non-pregnant women and pregnant women with and without recurrent miscarriage. Cells were cultured in vitro and stimulated with PHA. Numbers in brackets refer to range
 
Interestingly, differences were observed in cytokine production, which were subsequently reflected in pregnancy outcome (Figure 1Go). The level of TNF-{alpha} produced by pregnant women was significantly lower than for non-pregnant controls (P = 0.006), (Table IGo, Figure 1aGo). This reduction was more marked in RPL patients (P = 0.0003, Table IGo). Whereas RPL women with a successful reproductive outcome had similar concentrations of TNF-{alpha} to pregnant women, RPL women who subsequently miscarried had significantly lower levels than either pregnant (P = 0.02) or non-pregnant controls (P = 0.0004).





View larger version (30K):
[in this window]
[in a new window]
 
Figure 1. Scatter plot comparing cytokine production by peripheral blood mononuclear cells stimulated in vitro with PHA in RPL women whose pregnancy subsequently failed (RPL/Misc) or was successfully completed (RPL/LB), compared with normal pregnant women and non-pregnant controls. (a) TNF-{alpha}; (b) IFN-{gamma}; (c) IL-10. Bars refer to median values for each group.

 
Production of IFN- {gamma} was lower in pregnant than in non-pregnant women and even lower in RPL pregnant women. Statistical significance was reached when comparing RPL patients and non-pregnant controls (P = 0.0191, Table IGo). There was no difference in IFN- {gamma} between RPL patients who subsequently miscarried and those who subsequently had a live birth (Figure 1bGo). Detectable levels of IL-12 were found in only three RPL patients and three controls (data not shown).

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 IGo). Similar levels of IL-10 were found in RPL patients who subsequently miscarried, compared with RPL patients who subsequently had a live birth (Figure 1cGo).

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 20–337; 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 20–337) compared to those who had a live birth (n = 9; median 100 pg/ml, range 75–190).

One patient was sampled twice. The first sample was taken at eight weeks gestation when a fetal heartbeat was detected. At this time IFN- {gamma} and TNF-{alpha} 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- {gamma} was reduced by 90% and IL-10 was reduced by 50%. TNF-{alpha} levels were unchanged.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It has been proposed that during pregnancy, systemic maternal immune responses are biased in favour of a Th2 cytokine profile (Wegmann et al., 1993Go). Our data provides evidence to support this hypothesis and confirms published data that suggests that this is also the case in humans during normal pregnancy (Marzi et al., 1996Go; Makhseed et al., 1999Go; Raghupathy et al., 1999Go, 2000Go). However, our hypothesis that a failure of this Th1 to Th2 cytokine shift occurs in RPL and precedes a miscarriage in some of these women was not supported; on the contrary, a Th2 profile may be exacerbated.

In this study TNF-{alpha} 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-{alpha} 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-{alpha} 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., 1997Go). TNF-{alpha} production in late pregnancy is implicated in the induction of labour in mice (Chaout et al., 1990) and humans (Vince et al., 1992Go). Placental production of soluble receptors has been shown to modulate TNF-{alpha} function in pregnancy (Austgulen et al., 1992Go).

IFN- {gamma} 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- {gamma} 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., 1996Go) 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., 1994Go). 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., 1996Go; Tranchot-Diallo et al., 1997Go). 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-{alpha} 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., 1999Go; Raghupathy et al., 1999Go, 2000Go). 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 Th1–Th2 shift when compared with normal pregnant women, and the second (after the fetus had died) an apparent 50–90% 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., 1996Go). The pattern of cytokine production varies throughout pregnancy (Marzi et al., 1996Go; Tranchot-Diallo et al., 1997Go) 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., 1999Go).

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., 1996Go). 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., 1996Go; Lim et al., 1998Go), and in pregnancy a 10-fold increase in decidual Th2 cytokine secretion occurred (Krasnow et al., 1996Go). 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., 2000Go). In RPL women who had miscarried, more TNF-{alpha} was found in the decidua (Vives et al., 1999Go) but less in the trophoblast (Lea et al., 1997Go).

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., 1998Go). 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- {gamma} and reduced IL-10 and IL-4 when stimulated with trophoblast antigens (Hill et al., 1995Go). 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., 1999Go). 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., 1992Go; Perrey et al., 1999Go; Pravica et al., 1999aGo,bGo). However, these polymorphisms have not been found to be more prevalent amongst women with RPL (Babbage et al., 2001Go; Reid et al., 2001Go).

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by the NHS Executive Northwest, Research and Development Directorate.


    Notes
 
4 To whom correspondence should be addressed at Department of Immunology, Duncan Building, Daulby Street, Liverpool L69 3GA, UK. E-mail: g.s.vince{at}liverpool.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Austgulen, R., Liabakk, N.B., Brockhaus, M., and Espevik, T. (1992) Soluble TNF receptors in amniotic fluid and in urine from pregnant women. J. Reprod. Immunol., 22, 105–116.[ISI][Medline]

Babbage, S.J., Arkwright, P.D., Vince, G.S., Perrey, C., Pravica V., Quenby, S., Bates, M. and Hutchinson, I.V. (2001) Cytokine promoter gene polymorphisms and idiopathic recurrent pregnancy loss. J. Reprod. Immunol., 51, 21–27.[ISI][Medline]

Chaouat, G., Menu, E., Clark, D.A., Dy, M., Minkowski, M. and Wegmann, T.G. (1990) Control of fetal survival in CBA/JxDBA/2 mice by lymphokine therapy. J. Reprod. Fertil., 89, 447–453.[Abstract]

Da Salva, J.A. and Spector, T.D. (1992) Rheumatoid arthritis in pregnancy. Clin. Rheumatol., 11, 189–202.[ISI][Medline]

Delassus, S., Coutinho, G.C., Saucier, C., Darche, S. and Kourilsky, P. (1994) Differential cytokine expression in maternal blood and placenta during gestation. J. Immunol., 152, 2411–2420.[Abstract/Free Full Text]

Drakeley, A.J., Quenby, S. and Farquharson, R.G. (1998) Mid-trimester loss – appraisal of a screening protocol. Hum. Reprod., 13, 1975–1980.[Abstract]

Ecker, J.L., Laufer, M.R. and Hill, J.A. (1993) Measurement of embryotoxic factors is predictive of pregnancy outcome in women with a history of RSA. Obstet. Gynecol., 81, 84–87.[Abstract]

Hart, C.A. (1988) Pregnancy and host resistance. Baillière's Clin. Immunol. Allergy, 2, 735–757

Heyborne, K.D., McGregor, J.A., Henry, G., Witkin, S.S. and Abrams, J.S. (1995) IL-10 in amniotic fluid at midtrimester in relation to fetal growth. Am. J. Obstet. Gynecol., 171, 55–59.[ISI]

Hill, J.A., Polgar, K., Harlow, B.L. and Anderson, D.J. (1992) Evidence of embryo and trophoblast toxic cellular immune responses in women with RSA. Am. J. Obstet. Gynecol., 166, 1044–1052.[ISI][Medline]

Hill, J.A., Polgar, K. and Anderson, D.J. (1995) T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA, 273, 1933–1936.[Abstract]

Jenkins, C., Roberts, J., Wilson, R., MacLean, M.A., Shilito, J. and Walker, J.J. (2000) Evidence of a Th1 type response associated with recurrent miscarriage. Fertil. Steril., 73, 1206–1208.[ISI][Medline]

Krasnow, J.S., Tollerud, D.J., Naus, G. and DeLoia, J.A. (1996) Endometrial Th2 cytokine expression throughout the menstrual cycle and early pregnancy. Hum Reprod., 11, 1747–1754.[Abstract]

Lea, R.G., Tulppala, M. and Critchley, H.O. (1997) Deficient syncytiotrophoblast tumour necrosis factor-alpha characterizes failing first trimester pregnancies in a sub-group of recurrent miscarriage patients. Hum. Reprod., 12, 1313–1320.[ISI][Medline]

Lim, K.J.H., Odukoya, O.A., Li, T.C. and Cooke, I.D. (1996) Cytokines and immuno-endocrine factors in recurrent miscarriage. Hum. Reprod. Update, 2, 469–480.[Abstract/Free Full Text]

Lim, K.J.H., Odukoya, O.A., Ajjan, R.A., Li, T.C., Weetman, A.P. and Cooke I.D. (1998) Profile of cytokine mRNA expression in peri-implantation human endometrium. Mol. Hum. Reprod., 4, 77–81.[Abstract]

Lim, K.J.H., Odukoya, O.A., Ajjan, R.A. Li, T.C., Weetman, A.P. and Cooke, I.D. (2000) The role of T-helper cytokines in human reproduction. Fertil. Steril., 73,136–142.[ISI][Medline]

Marzi, M., Vigano, A., Trabattoni, D., Villa, M.L., Salvaggio, A., Clerici, E. and Clerici, M. (1996) Characterisation of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin. Exp. Immunol., 106, 127–133.[ISI][Medline]

Makhseed, M., Raghupathy, F., Azizeh, F., Al-Azemi, M.M., Hassan, N.A. and Bandar, A. (1999) Mitogen-induced cytokine responses of maternal peripheral blood lymphocytes indicate a differential Th-type bias in normal pregnancy and pregnancy failure. Am. J. Reprod. Immunol., 42, 273–281.[ISI][Medline]

Müller-Eckhardt, G., Mallmann, P., Neppert, J., Lattermann, A., Melk, A., Heine, O., Pfeiffer, R., Zingsem, J., Domke, N. and Mohr-Pennert, A. (1994) Immunogenetic and serological investigations in nonpregnant and in pregnant women with a history of RSA. J. Reprod. Immunol., 27, 95–109.[ISI][Medline]

Perrey, C., Turner, S.J., Pravica, V., Howell, W.M. and Hutchinson, I.V. (1999) ARMS-PCR methodologies to determine IL-10, TNF-{alpha}, TNF-ß and TGF-ß1 gene polymorphisms. Transpl. Immunol., 7, 127–1278.[ISI][Medline]

Piccinni, M.P., Giudizi, M.G., Biagiotti, R., Beloni, L., Giannarini, L., Sampognaro, S., Parronchi, P., Manetti, R., Annunziato, F., Livi, C. et al. (1995) Progesterone favours the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. J. Immunol., 155, 129–133.

Piccinni, M.P. and Romagnani, S. (1996) Regulation of fetal allograft survival by hormone-controlled Th1- and Th2-type cytokines. Immunol. Res., 15, 141–150.[ISI][Medline]

Piccinni, M.P., Beloni, L., Livi, C., Maggi, E., Scarselli, G. and Romagnani, S. (1998) Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat. Med., 4, 1020–1024.[ISI][Medline]

Pravica, V., Asderakis, A., Perrey, C., Hajeer, A., Sinnott, P.J. and Hutchinson, I.V. (1999a) In vitro production of IFN-gamma correlates with CA repeat polymorphism in the human IFN-gamma gene. Eur. J. Immunogenet., 26, 1–3.[ISI][Medline]

Pravica, V., Perrey, C., Brogan, I.J. and Hutchinson, I.V. (1999b) A single nucleotide polymorphism correlates with a polymorphic CA microsatellite in the first intron of the human IFN-{gamma} gene. Immunology,98, (Suppl. 1) 163.

Quenby, S. and Farquharson, R. (1993) Predicting recurring miscarriage: What is important? Obstet. Gynecol., 82, 132–138.[Abstract]

Quenby, S., Bates, M., Doig, T., Brewster, J., Lewis-Jones, D.I., Johnson, P.M. and Vince, G. (1999) Pre-implantation endometrial leucocytes in women with recurrent miscarriages. Hum. Reprod., 14, 737–741.

Robertson, S.A., Seamark, R.F., Guilbert, L.J. and Wegmann, T.G. (1994) The role of cytokines in gestation. Crit. Rev. Immunol., 14, 239–292.[ISI][Medline]

Raghupathy, R., Makseed, M., Azizieh, F., Hassan, N., Al-Azemi, M. and Al-Shamali, E. (1999) Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell. Immunol., 196, 122–130.[ISI][Medline]

Raghupathy, R., Makseed, M., Azizieh, F., Omu, A., Gupta, M. and Farhat, R. (2000) Cytokine production by maternal lymphocytes during normal pregnancy and in unexplained recurrent spontaneous abortion. Hum. Reprod., 15, 713–718.[Abstract/Free Full Text]

Reid, J.J., Simpson, N.A., Walker, R.G., Economidou, O., Shillito, J., Gooi, H.C., Duffy, S.R. and Walker, J.J. (2001) The carriage of pro-inflammatory cytokine gene polymorphisms in recurrent pregnancy loss. Am. J. Reprod. Immunol., 45, 35–40.[ISI][Medline]

Tranchot-Diallo, J., Gras, G., Parnet-Mathieu, F., Benveniste, O., Marce, D., Roques, P., Milliez, J., Chaouat, G. and Dormont, D. (1997) Modulations of cytokine expression in pregnant women. Am. J. Reprod. Immunol., 37, 215–216.[ISI][Medline]

Vince, G.S. and Johnson, P.M. (1996) Is there a Th2 bias in human pregnancy? J. Reprod. Immunol., 32, 101–104.[ISI][Medline]

Vince, G.S., Shorter, S., Starkey, P., Humphreys, J., Clover, L., Wilkins, T., Sargent, I. and Redman, C. (1992) Localization of tumour necrosis factor production in cells at the materno/fetal interface in human pregnancy. Clin. Exp. Immunol., 88, 174–180.[ISI][Medline]

Vives, A., Balasch, J., Yague, J., Quinto, L., Ordi, J. and Vanrell, J.A. (1999) Type-1 and type-2 cytokines in human decidual tissue and trophoblasts from normal and abnormal pregnancies detected by reverse transcriptase polymerase chain reaction (RT-PCR). Am. J. Reprod. Immunol., 42, 361–368.[ISI][Medline]

Wegmann, T.G., Lin, H., Guilbert, L. and Mosmann, T.R. (1993) Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a Th2 phenomenon? Immunol. Today, 14, 353–356.[ISI][Medline]

Wilson, A.G., di Giovine, F.S., Blakemore, M. and Duff, G.W. (1992) Single base polymorphism in the tumour necrosis factor-alpha (TNF-alpha) gene detectable by NcoI restriction of the PCR product. Hum. Mol. Genet., 1, 353.[Medline]

Yamada, H., Polgar, K. and Hill, J.A. (1994) Cell-mediated immunity to trophoblast antigens in women with RSA. Am. J. Obstet. Gynecol., 170, 1339–1344.[ISI][Medline]

Submitted on May 10, 2001; accepted on May 3, 2002.