Modulation of cytokine production by dydrogesterone in lymphocytes from women with recurrent miscarriage

Transcription

1 BJOG: an International Journal of Obstetrics and Gynaecology August 2005, Vol. 112, pp DOI: /j x Modulation of cytokine production by dydrogesterone in lymphocytes from women with recurrent miscarriage Raj Raghupathy, a Esraa Al Mutawa, a Ma asoumah Makhseed, b Fawaz Azizieh, a Julia Szekeres-Bartho c Objective To examine the effects of dydrogesterone on the production of Th1 and Th2 cytokines by lymphocytes from women undergoing unexplained recurrent spontaneous miscarriage (RSM). Design Controlled prospective, clinical study conducted in a maternity hospital and a university-based immunology laboratory. Setting Faculty of Medicine, Kuwait University and Kuwait Maternity Hospital. Sample Thirty women with unexplained RSM. Methods Peripheral blood mononuclear cells (PBMC) from women with unexplained RSM were isolated from venous blood by density gradient sedimentation and stimulated with phytohaemagglutinin (PHA). Culture supernatants assayed for interferon (IFN)-g, tumour necrosis factor (TNF)-a, interleukin (IL)-4, IL-6 and IL-10 by ELISA. Levels of the progesterone-induced blocking factor (PIBF) were also measured. Main outcome measures Cytokine production in the presence and absence of progesterone and dydrogesterone. Results Dydrogesterone significantly inhibited the production of the Th1 cytokines IFN-g (P ¼ ) and TNF-a (P ¼ 0.005) and induced an increase in the levels of the Th2 cytokines IL-4 (P ¼ 0.03) and IL-6 (P ¼ 0.017) resulting in a substantial shift in the ratio of Th1/Th2 cytokines. The effect of dydrogesterone was blocked by the addition of the progesterone-receptor antagonist mifepristone, indicating that dydrogesterone was acting via the progesterone receptor. Dydrogesterone induced the production of PIBF. Conclusion Dydrogesterone inhibits the production of the Th1 cytokines IFN-g and TNF-a from lymphocytes and up-regulates the production of the Th2 cytokines IL-4 and IL-6, inducing a Th1 to Th2 cytokine shift. INTRODUCTION Cytokines associated with the cellular immune response may be responsible for at least a proportion of unexplained recurrent spontaneous miscarriages (RSMs), 1,2 which accounts for about 40 60% of all cases of RSMs. 3 It has been suggested that successful pregnancy is associated with a down-regulation of Th1-type activity and enhancement of Th2-type activity. 1,2,4 6 Interleukin (IL)-2, interferon (IFN)-g and tumour necrosis factor (TNF) are cytokines with adverse effects on the conceptus and are characteristic of Th1-type T cells. These cytokines induce cytotoxic and inflammatory reactions and are responsible for the induction of cell-mediated inflammation. 7 Th2 cells secrete IL-4, a Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait b Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University, Kuwait c Department of Medical Microbiology and Immunology, Pecs University Medical School, Hungary Correspondence: Dr R. Raghupathy, Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, 13110, Kuwait. D RCOG 2005 BJOG: an International Journal of Obstetrics and Gynaecology IL-5, IL-6, IL-10 and IL-13, which are associated with the induction of humoural immunity. Some of these cytokines have anti-inflammatory activity. 7 Elevated levels of the Th1 cytokines IL-2 and IFN-g, and decreased levels of the Th2 cytokine IL-10, have been reported after antigen- and mitogen-induced activation of peripheral blood mononuclear cells (PBMC) from women with spontaneous recurrent miscarriage. 8 The activation of PBMC with trophoblast antigens has shown that women with unexplained RSM have a Th1-type cytokine profile. 1 We have previously demonstrated increased production of IFN-g, TNF-a, TNF-h and IL-2 by mitogen-activated PBMC from women with RSM and 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. 9 A similar Th1 bias in unexplained RSM and a Th2 bias in successful pregnancy is seen after stimulation with autologous placental cells and with trophoblast antigens. 10 Piccinni et al. 11 demonstrated significantly greater numbers of IL-4- and IL-10-producing T cell clones generated from cells infiltrating the decidua of women with normal pregnancy compared with those undergoing unexplained RSM. These data support the contention that women with normal pregnancy have a higher Th2 bias, while women with a history of unexplained RSM have a bias towards Th1 reactivity.

2 CYTOKINE MODULATION BY DYDROGESTERONE 1097 However, the association between Th1/ Th2 cytokines and pregnancy outcome has been challenged by some researchers. Bates et al. 12 reported that the production of IL-4 and IL-10 was increased in women with a history of recurrent pregnancy loss while no differences were observed in the levels of the Th1 cytokine IFN-g. Shimada et al. 13 reported higher levels of IL-4-positive T cells on flow cytometry analysis in women with RSM, suggesting increased levels of Th2-type cells in these women. This group has also reported that treatment of women with RSM with intravenous immunoglobulin leads to decreased Th1/ Th2 lymphocyte ratios and suggest that high Th1/ Th2 ratios in RSM may have an aetiological basis. 14 Based on the demonstration of both pro- and anti-inflammatory cytokines at the uteroplacental interface in mice and the beneficial roles for pro-inflammatory cytokines such as IFN-g, Zourbas et al. 15 suggested that influences of Th1 and Th2 cytokines are complex and probably occur in sequential windows. Despite these conflicting data, substantial literature exists to suggest 1,8 11,16 20 an increased Th1-bias in recurrent miscarriage. This possible association between RSM and maternal Th1 cytokine bias raises the possibility of manipulating the Th1/Th2 cytokine balance to down-regulate Th1 cytokines and increase the chances of a successful pregnancy. Progesterone, which has anti-inflammatory and immunosuppressive properties, 21 favours the development of human T cells producing Th2 cytokines. 22 Progesterone also blocks the production of Th1 cytokines by lymphocytes responding to in vitro stimulation by a trophoblast antigen extract. 23 In this study, we have investigated the potential immunomodulatory properties of dydrogesterone. Dydrogesterone is a potent orally administered progestogen, similar to endogenous progesterone in its molecular structure and pharmacological effects, with a high affinity for the progesterone receptor. We also studied the effect of dydrogesterone on the production of progesterone-induced blocking factor (PIBF), which is a protein produced by pregnancy lymphocytes following exposure to progesterone. 24 PIBF inhibits several cell-mediated immune responses particularly cell-mediated cytotoxicity and natural killer (NK) cell activity and is thought to play an important immunoprotective role in pregnancy. 25 METHODS This study was approved by the Ethics Committees of the Faculty of Medicine, Kuwait University and Kuwait Maternity Hospital. Thirty women with a history of at least three RSMs in the first trimester and no history of successful pregnancy were enrolled into the study. Patients were routinely investigated for possible anatomical, endocrinologic, infectious, genetic and immunological causes of miscarriage; immunological tests included lupus anticoagulant, anti-cardiolipin and anti-nuclear antibodies. These subjects had no demonstrable cause for their pregnancy losses. Informed consent was obtained from women who volunteered to participate, before blood samples were collected. Peripheral blood was obtained by venipuncture from subjects on the day of miscarriage. PBMC were separated by Ficoll-paque (Pharmacia Biotech, Sweden) density gradient centrifugation, suspended in RPMI medium (GIBCOBRL, Gaithersburg, USA) containing 10% fetal calf serum (GIBCOBRL), aliquoted into 96-well tissue culture plates at a density of 10 5 cells per well and then challenged with the mitogen phytohaemagglutinin (PHA) (Sigma- Aldrich, St Louis, Missouri, USA) at a concentration of 5 Ag/mL for a period of 96 hours. Supernatants were harvested at 96 hours for the estimation of cytokine levels. The optimal concentrations of progesterone (Sigma- Aldrich) and dydrogesterone (provided by Solvay Pharmaceuticals, Hannover, Germany) were determined during standardisation experiments. In initial standardisation experiments on 10 samples, progesterone and dydrogesterone were tested at three concentrations 10 4 mol/l, 10 5 mol/l and 10 6 mol/l. While dydrogesterone and progesterone at 10 4 mol/l showed inhibitory effects on the production of IFN-g (P ¼ 0.001), this dose did not affect the production of TNF-a. At the lowest concentrations of 10 6 mol/l dydrogesterone and progesterone, no effect was observed on the production of IFN-g and TNF-a. Dydrogesterone and progesterone at 10 5 mol/l brought about significant reduction in the levels of IFN-g (P ¼ for dydrogesterone, P ¼ 0.02 for progesterone) and TNF-a (P ¼ for dydrogesterone, P ¼ 0.05 for progesterone) (data not shown). Similarly, at 10 6 mol/l, progesterone did not Table 1. Cytokine production in response to mitogen and progestogens. Values are presented as mean [SEM]. Cytokine PHA (pg/ml) PHA þ dydrogesterone (pg/ml) PHA þ progesterone (pg/ml) P* P** IFN-g 47,669 [3960] 4810 [736] 37,279 [4412] TNF-a 5460 [507] 3798 [676] 3785 [385] IL [2.7] 34.4 [6.4] 29.7 [4.3] Il-6 11,220 [834] 14,237 [845] 13,540 [745] IL [107] 918 [132] 1128 [149] * Values for PHA compared with PHA þ dydrogesterone. ** Values for PHA compared with PHA þ progesterone.

3 1098 R. RAGHUPATHY ET AL. Table 2. Cytokine ratios before and after addition of progestogen to stimulated cultures. Cytokine ratio PHA PHA þ dydrogesterone PHA þ progesterone P* P** IFN/IL IFN/IL IFN/IL TNF/IL TNF/IL TNF/IL * Values for PHA compared with PHA þ dydrogesterone. ** Values for PHA compared with PHA þ progesterone. induce increased production of the Th2 cytokines IL-4, IL-6 and IL-10 (data not shown). Dydrogesterone and progesterone at 10 5 mol/l induced significant increase in the production of IL-4 (P ¼ for dydrogesterone, P ¼ for progesterone) and IL-6 (P ¼ for dydrogesterone, P ¼ for progesterone). Based on this dose response analysis we chose the concentration of 10 5 mol/l of dydrogesterone and progesterone for further studies. Each hormone was tested by addition to the culture wells in addition to the presence of PHA described above. In order to verify whether the immunomodulatory effects of dydrogesterone and progesterone are mediated via the progesterone receptor, the progesterone receptor antagonist mifepristone (Sigma-Aldrich) was also added to the supernatant. In initial standardisation experiments, samples from four subjects were studied. Mifepristone at 10 3,10 4,10 5, 10 6 and 10 7 mol/l was added to PHA-stimulated PBMC cultures, and cytokine production then assessed. Based on these experiments, the dose of 10 6 mol/l of mifepristone was selected because mifepristone by itself at doses of 10 3, 10 4 and 10 5 brought about a reduction in levels of IFN-g and TNF-a. At10 6 mol/l, mifepristone did not affect cytokine production by itself nor does it affect cell proliferation (data not shown). Cytokine levels were measured in supernatants using antibody-sandwich ELISA kits (Coulter Immunotech, Marseilles, France) according to the manufacturer s instructions. Standard curves were plotted for each of the cytokines tested using reference cytokines and the results read from these curves. All samples and standards were tested in triplicate. Intra- and inter-assay variability were both within acceptable limits, with coefficient of variation values <10. The sensitivities of detection of cytokines are as follows: 3 pg/ml of IFN-g, 10 pg/ml of TNFa, 5 pg/ml of IL-4, 3 pg/ml of IL-6 and 5 pg/ml of IL-10. For the estimation of the PIBF, PBMC were obtained from pregnant women, aliquoted at cells per ml in tissue culture microtiter plates and treated with different concentrations of dydrogesterone ( Ag/mL) in the presence of mifepristone for 16 hours at 37 C in a humid chamber. Lymphocytes were washed in order to eliminate excess hormones and centrifuged onto microscope glass slides, which were then fixed with cold acetone and then incubated with 1:100 diluted monoclonal anti-pibf antibodies, for 1 hour. The slides were washed twice with PBS and incubated with 1:100 diluted horseradish peroxidasecoupled anti-mouse IgG (Sigma) for 30 minutes. The reaction was developed with aminoethylcarbazole. Nuclei were counterstained with haematoxylin and the slides mounted with gelatin glycerol. The percentage of PIBF-positive cells was determined by counting under the microscope. The standard Mann Whitney U test was used for nonparametric comparisons of stimulation indices and median cytokine levels, as the data were not normally distributed. Differences were considered significant if the P value RESULTS The presence of dydrogesterone or progesterone in PBMC cultures resulted in significantly reduced levels of the Th1 cytokines, IFN-g and TNF-a (Table 1). Levels of both IL-4 and IL-6 were significantly elevated in the presence of dydrogesterone or progesterone, but IL-10 production was unaffected (Table 1). Because relative levels of Th1 and Th2 cytokines are probably of greater importance than their absolute levels, we calculated the ratios of Th1 to Th2 cytokines. A marked reduction in these ratios was observed in cultures containing dydrogesterone or progesterone indicating a decrease in Th1 cytokine bias (Table 2). Table 3. The effect of mifepristone on progestogen-induced changes in cytokine ratios. Cytokine ratio PHA þ dydrogesterone PHA þ mifepristone þ dydrogesterone P * PHA þ progesterone PHA þ mifepristone þ progesterone P ** IFN/IL IFN/IL IFN/IL TNF/IL TNF/IL TNF/IL * Values for PHA þ dydrogesterone compared with PHA þ dydrogesterone þ mifepristone. ** Values for PHA þ progesterone compared with PHA þ progesterone þ mifepristone.

4 Table 4. Percentage of cells staining for PIBF after incubation with increasing concentration of dydrogesterone. Mitogen-simulated PBMC from 15 subjects with unexplained RSM were cultured with dydrogesterone or progesterone in the presence or absence of 10 6 mol/l of mifepristone. mifepristone blocked the observed increase in the levels of IFN-g and TNF-a, and the observed decrease in the levels of IL-4 and IL-6. While dydrogesterone and progesterone brought about a reduction in Th1 Th2 cytokine ratios, the addition of mifepristone in these cultures results in increased Th1 Th2 cytokine ratios (Table 3). Dydrogesterone induced the production of PIBF in a dose-dependent manner (Table 4) with 100 mg/ml of dydrogesterone bringing about an almost fourfold increase in PIBF production. The addition of mifepristone to these cultures resulted in an almost complete inhibition of PIBF production; while the addition of dydrogesterone to lymphocyte cultures increased the PIBF-positivity from 21% to 5%, the presence of mifepristone in the cultures results in a reversal of PIBF positivity to 22% (data not shown). DISCUSSION Concentration of dydrogesterone (AMg/mL) % PIBF-positive cells Evidence for a dichotomous maternal T-helper reactivity in pregnancy first emerged from animal studies, which showed that normal pregnancy is associated with a protective Th2 cytokine pattern, while pregnancy loss can be mediated by a maternal Th1 bias. 4,26 In humans, flow cytometry studies have shown that normal pregnancy is associated with increased levels of IL-4-producing T cells and reduced levels of IFN-g- and IL-2-producing cells indicating a shift towards Th2 bias. 27 While a direct cause-and-effect relationship between maternal Th1 reactivity and human pregnancy loss has not been established, the maternal cytokine bias is skewed in women with unexplained RSM; higher levels of Th1 cytokines are produced by mitogen- or antigen-activated PBMC from recurrent aborters while higher levels of Th2 cytokines are secreted by pregnant women with a history of reproductive 1,8 11,16 20 success. We have demonstrated that dydrogesterone downregulates Th1 cytokines and up-regulates Th2 cytokines resulting in a shift in cytokine bias away from Th1 dominance and towards Th2-bias. IFN-g and TNF-a have been shown to deter embryo development, implantation events and proliferation of the trophoblast cells 26 and to have apoptotic effects on human trophoblast cells. 27 In animals, these cytokines bring about fetal demise when injected during CYTOKINE MODULATION BY DYDROGESTERONE 1099 gestation 28 and up-regulate the procoagulant, fg IFN-g and TNF-a may also mediate placental/fetal damage via activation of NK activity and macrophages. IL-4 is the quintessential Th2 cytokine and increased production of IL-4 has been shown to favour further Th2 bias. 7 Progesterone has been shown to promote the differentiation of T cells into Th2 effectors and is thought to be responsible for a Th2 switch at the maternal fetal interface during normal, successful gestation. 22 In this study, progesterone was tested at a concentration (10 5 mol/l) that is similar to that achieved at maternal fetal tissues. 24 Thus, the increased production of the Th2 cytokine IL-4 and the decreased production of the Th1 cytokines IFN-g and TNF-a together could well result in a substantial swing in Th1/Th2 reactivity towards the pregnancy-conducive Th2 profile and away from the potentially harmful Th1 profile. PIBF is a protein produced by pregnancy lymphocytes upon exposure to progesterone that inhibits various cellmediated immune reactions. 24,25 In mice 30 and in humans (Raghupathy et al., unpublished data) PIBF up-regulates Th2 cytokine production and thus brings about a shift from Th1 to Th2 cytokine bias. The percentage of PIBFpositive lymphocytes is significantly reduced in women prone to pregnancy loss, preterm labour and threatened miscarriage. 31 PIBF prevents fetal resorptions in a murine model of pregnancy loss 32 and is thought to play an important role in pregnancy by helping the conceptus escape immune surveillance. 24,30 Thus, the ability of dydrogesterone to induce the production of PIBF may well provide an indirect beneficial effect in addition to its ability to directly effect a Th1 to Th2 cytokine shift. Our data from co-incubation with mifepristone suggest that progesterone and dydrogesterone induce cytokine modulation by binding to the progesterone receptor on lymphocytes. We cannot rule out the possibility that they may act via the glucocorticoid receptor, although a previous study demonstrated that the inhibition of PIBF production was blocked by mifepristone but not by a specific glucocorticoid receptor blocker (RU43044). 33 Blood samples for this study were obtained at the time of miscarriage and it has been demonstrated that the timing of the sampling could influence cytokine profiles. 19,34 Marzi et al. 8 have reported increased production of Th1 cytokines in women who aborted, when samples were taken prior to miscarriage. We have previously demonstrated that miscarriage-prone women who undergo another spontaneous miscarriage have a stronger Th1-bias than miscarriageprone women who proceed to have a normal delivery. 18 However, cytokine profiles do need to be established at different time points during gestation for us to ascertain Th1 versus Th2 bias at different stages of pregnancy. Choi et al. 23 suggest that potentially immunosuppressive doses of progesterone may benefit individuals in whom the aetiology of RSM is related to predominance of maternal Th1 cytokines. However, orally administered progesterone is poorly absorbed, is subject to first-pass mechanism, has a

5 1100 R. RAGHUPATHY ET AL. short biologic half life, 35 loses much of its bioactivity 36 and is rapidly cleared. 37 The progesterone used in this study is synthetically prepared from a naturally occurring plant steroid precursor and not the micronised version currently available for oral and vaginal use. Upon oral administration, the micronised form is rapidly metabolised into nonprogestogenic metabolites due to the first-pass effect. 37,38 There is also considerable intraindividual and interindividual variation in the metabolism of orally administered micronised progesterone. 39 On the other hand, the high bioavailability of orally administered dydrogesterone makes it attractive from a potentially therapeutic perspective. This study is based on in vitro experiments; if clinical trials confirm the cytokine-redirecting capability of dydrogesterone, it may offer the potential for an effective, safe and orally administered therapeutic intervention in RSM with high Th1 cytokine bias as the aetiology. CONCLUSION Dydrogesterone has an immunomodulatory capability and appears to induce a maternal cytokine shift from Th1 cytokine dominance towards a Th2 bias. This may act directly and also indirectly via the production of the PIBF. Acknowledgments Supported by Grant MI05/02 from Kuwait University Research Administration. The authors thank Mr Parvez Rawoot for technical support. References 1. Hill JA, Anderson DJ, Polgar K. T helper 1-type cellular immunity to trophoblast in women with recurrent spontaneous abortions. JAMA 1995;273: Raghupathy R. Pregnancy: success and failure within the Th1/Th2/ Th3 paradigm. Semin Immunol 2001;13: Stray-Pederson B, Stray-Pederson S. Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984;148: Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal fetal relationship: is successful pregnancy a Th2 phenomenon? Immunol Today 1993;14: Guilbert LJ. There is a bias against type 1 (inflammatory) cytokine expression and function in pregnancy. J Reprod Immunol 1996;32: Chaouat G, Cayol V, Mairovitz V, Dubanchet S. Localization of the Th2 cytokines IL-3, IL-4, IL-10 at the murine fetomaternal interface during pregnancy. In: Gupta SK, editor. Reproductive Immunology. New Delhi: Narosa Publishing House, 1999: Mosmann TR, Sad S. The expanding universe of T-cell subsets. Immunol Today 1996;17: Marzi M, Vigano A, Trabattoni D, et al. Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin Exp Immunol 1996;106: Raghupathy R, Makhseed M, Azizieh F, Omu A, Gupta M, Farhat R. Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion. Hum Reprod 2000;15: Raghupathy R, Makhseed M, Azizieh F, Hassan N, Al-Azemi M, Al- Shamali E. Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell Immunol 1999;196: Piccinni M-P, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factor and type 2 T- helper cytokines by decidual T cells in unexplained recurrent abortions. Nature (Med) 1998;4: Bates MD, Quenby S, Takakuwa K, Johnson PM, Vince GS. Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss? Human Reprod 2002;17: Shimada S, Iwabuchi K, Kato EH, et al. No difference in naturalkiller-t cell population but Th2/Tc2 predominance in peripheral blood of recurrent aborters. Am J Reprod Immunol 2003;50: Yamada H, Morikawa M, Furuta I, et al. Intravenous immunoglobulin treatment in women with recurrent abortions: increased cytokine levels and reduced Th1/Th2 lymphocyte ratio in peripheral blood. Am J Reprod Immunol 2003;49: Zourbas S, Dubanchet S, Martal J, Chaouat G. Localization of proinflammatory (IL-12, IL-15) and anti-inflammatory ( IL-11, IL-13) cytokines at the feto-maternal interface during murine pregnancy. Clin Exp Immunol 2001;126: Jenkins C, Roberts J, Wilson R, MacLean MA, Shilito J, Walker JJ. Evidence of a Th1 type response associated with recurrent miscarriage. Fertil Steril 2000;73: Piccinni MP, Scaletti C, Vultaggio A, Maggi E, Romagnani S. Defective production of LIF, M-CSF and Th2-type cytokines by T cells at fetomaternal interface is associated with pregnancy loss. J Reprod Immunol 2001;52: Makhseed M, Raghupathy R, Azizieh F, Al-Shamali E, Ashkanani L. Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions. Hum Reprod 2001;16: Laird SM, Tuckerman EM, Cork BA, Linjawi S, Blakemore AI, Li TC. A review of immune cells and molecules in women with recurrent miscarriage. Hum Reprod Update 2003;9: Lim KJ, Odukoya OA, Ajjan RA, Li TC, Weetman AP, Cooke ID. The role of T-helper cytokines in human reproduction. Fertil Steril 2000;73: Stites DP, Bugbee S, Siiteri PK. Differential actions of progesterone and cortisol on lymphocyte and monocyte interaction during lymphocyte activation relevance to immunosuppression in pregnancy. J Reprod Immunol 1983;5: Piccinni MP, Giudizi MG, Biagiotti R, et al. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established T cell clones. J Immunol 1995;155: Choi BC, Polgar K, Xiao L, Hill JA. Progesterone inhibits in vitro embryotoxic Th1 cytokine production to trophoblast in women with recurrent pregnancy loss. Hum Reprod 2000;15(Suppl 1): Szekeres-Bartho J. Immunosuppression by Progesterone in Pregnancy. Boca Raton: CRC Press, Szekeres-Bartho J, Par G, Dombay G, Smart CY, Volgyi Z. The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity. Cell Immunol 1997;177: Haimovici F, Hill JA, Anderson DJ. The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro. Biol Reprod 1991;44: Yui J, Garcia-Lloret M, Wegmann TG, Guilbert L. Cytotoxicity of tumor necrosis factor-a (TNF-a) and gamma-interferon (IFN-g) against primary human placental trophoblasts. Placenta 1994;15:

6 CYTOKINE MODULATION BY DYDROGESTERONE Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBAxDBA/2 mice by lymphokine therapy. J Reprod Fertil 1990;89: Knackstedt M, Ding JW, Arck PC, et al. Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia. Am J Reprod Immunol 2001;46: Szekeres-Bartho J, Wegmann TG. A progesterone-dependent immunomodulatory protein alters the Th1/ Th2 balance. J Reprod Immunol 1996;31: Szekeres-Bartho J, Faust Z, Varga P. The expression of a progesteroneinduced immunomodulatory protein in pregnancy lymphocytes. Am J Reprod Immunol 1995;34: Szekeres-Bartho J, Chaouat G. Lymphocyte-derived, progesteroneinduced blocking factor corrects resorption in a murine abortion system. Am J Reprod Immunol 1990;23: Szekeres-Bartho J, Philibert D, Chaouat G. Progesterone suppression of pregnancy lymphocytes is not mediated by glucocorticoid effect. Am J Reprod Immunol 1990;23: Chaouat G, Ledee-Bataille N, Dubanchet S, Zourbas S, Sandra O, Martal J. Th1/ Th2 paradigm in pregnancy: paradigm lost? Int Arch Allergy Immunol 2004;134: Stanczyk FZ. Pharmacokinetics of progesterone administered by the oral and parenteral routes. J Reprod Med 1999;44: Simon JA, Robinson DE, Andrews MC, et al. The absorption of oral micronised progesterone: the effect of food, dose proportionality and comparison with intramuscular progesterone. Fertil Steril 1993;60: Maxson WS, Hargrove JT. Bioavailability of oral micronized progesterone. Fertil Steril 1985;44: Tavaniotou A, Smitz J, Bourgain C, Devroey P. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update 2000;6: McAuley JW, Kroboth FJ, Kroboth PD. Oral administration of micronized progesterone a review and more experience. Pharmacotherapy 1996;16: Accepted 4 January 2005