No difference in natural killer or natural killer T-cell population, but aberrant T-helper cell population in the endometrium of women with repeated miscarriage

Shigeki Shimada1, Emi Hirayama Kato1, Mamoru Morikawa1, Kazuya Iwabuchi2, Ryutaro Nishida1, Reiko Kishi3, Kazunori Onoé2, Hisanori Minakami1 and Hideto Yamada1,4

1 Deartment of Obstetrics and Gynecology, and 3 Department of Public Heath, Hokkaido University Graduate School of Medicine and 2 Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Graduate School of Medicine, Hokkaido University, Kita-ku N15 W7, Sapporo 060-8638, Japan. e-mail: yhideto{at}med.hokudai.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
BACKGROUND: The aim of this study was to assess the natural killer (NK) cell and natural killer T (NKT) cell populations and cytokine expression of T-helper (Th) cells in the endometrium of women who suffered from unexplained repeated miscarriage (RM). METHODS: The percentages of NK cells, NKT cells and CD4+ cells expressing intracellular interferon (IFN)-{gamma}, interleukin (IL)-4 and tumour necrosis factor (TNF)-{alpha} were measured by flow cytometry in the endometrium of 20 RM women and 17 fertile control women in the mid-luteal phase of the menstrual cycle. RESULTS: No significant differences in CD56+ NK cell or CD3+CD4CD8V{alpha}24+V{beta}11+ NKT cell percentages were found between RM and control women. However, in RM women compared with control women, the percentages of CD3+ cells (mean 40.3 versus 56.5%), CD4+IFN-{gamma}+ cells (28.4 versus 39.5%) and CD4+TNF-{alpha}+ cells (32.9 versus 45.8%) were significantly lower. The Th1/Th2 cell balance in RM women did not differ from that of controls. CONCLUSIONS: Immunodystrophism detected as diminution of the Th cell population rather than Th1 predominance, NK cell or NKT cell accentuation in the endometrium might underlie the pathophysiology of unexplained RM. This finding provokes an additional controversy on the Th1/Th2 balance concerning RM aetiology.

Key words: immunodystrophism/natural killer cell/natural killer T cell/recurrent miscarriage/Th cytokine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
Numerous investigations have been performed focusing on the possible role of immunological abnormalities in repeated miscarriage (RM). It has been found that peripheral natural killer (NK) cell activity and percentages prior to conception and during early pregnancy in RM women whose pregnancies are destined to end in miscarriage with normal fetal chromosome karyotype are higher than those in women with subsequent live birth and in women with subsequent miscarriage with abnormal karyotype (Yamada et al., 2001Go, 2003). These findings suggest that abnormal increases in peripheral NK cell parameters may be causally associated with RM.

An increasing number of unique functions of natural killer T (NKT) cells have been reported ever since this new lymphocyte was first reported in 1987 (Joyce, 2001Go). In the field of reproductive medicine, it has been found that NKT cells increase in the uterine decidua during early pregnancy (Dang and Heyborne, 2001Go), and might control the T-helper (Th) cell function by producing interferon (IFN)-{gamma} and interleukin (IL)-4 at the materno-fetal interface (Tsuda et al., 2001Go). However, no abnormality of the NKT cell population has yet been demonstrated in relation to the aetiology of RM. We previously found no difference in the NKT cell population of peripheral lymphocytes in the mid-luteal phase between RM women and fertile control women (Shimada et al., 2003Go).

The Th cell responses following activation are functionally characterized according to cytokine production; type-1 Th (Th1) and type-2 Th (Th2) cells (Mosmann and Coffman, 1989Go). The Th1 cells mainly secrete IL-2, IFN-{gamma} and tumour necrosis factor (TNF), whereas the Th2 cells synthesize IL-4, IL-5, IL-10 and IL-13. This Th1/Th2 balance model has been expected to provide a framework to explain materno-fetal immune reactions. Murine studies have demonstrated that the predominant Th1 immunity is related to implantation failure and fetal resorptions (Krishnan et al., 1996Go). The Th2 cytokines produced at the materno-fetal interface are thought to be beneficial for the maintenance of pregnancy because these suppress cellular cytotoxicity (Lin et al., 1993Go; Wegmann et al., 1993Go).

An abnormal Th1/Th2 balance with Th1 predominance of peripheral mononuclear cells in response to trophoblast antigens has been found to be associated with the cause of RM (Hill et al., 1995Go; Raghupathy et al., 1999Go). Recently, by using flow cytometry, one study found higher ratios of IFN-{gamma}+/IL-4+, TNF-{alpha}+/IL-4+ and TNF-{alpha}+/IL-10+ cells in CD3+CD8 peripheral lymphocytes in non-pregnant RM women (Kwak-Kim et al., 2003Go). However, the same study also found no differences in the percentages of IFN-{gamma}+, TNF-{alpha}+ or IL-4+ cells. In contrast, we demonstrated Th2 and Tc2 predominance of CD4+ and CD8+ peripheral lymphocytes in non-pregnant RM women (Shimada et al., 2003Go). Another study, using stimulated peripheral lymphocytes, found that TNF-{alpha} production levels at 6–10 weeks of gestation in RM women with subsequent miscarriages were lower than those in RM women with a successful reproductive outcome. The authors also found that IL-4 and IL-10 production levels at 6–10 weeks of gestation in RM women were higher than those in normal pregnant women (Bates et al., 2002Go). Thus, there still are many controversies concerning peripheral Th1/Th2 balance as the underlying pathophysiology and predictive values for miscarriage in RM women.

In the present study, we aimed to investigate the NK cell and NKT cell populations, the cytokine expression of lymphocyte, and the Th1/Th2 balances in the endometrium. The concept of immunodystrophism as the underlying pathophysiology of RM was proposed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
Patient and control characteristics
Twenty non-pregnant women with a history of primary RM and unexplained aetiology (mean ± SD, 33.1 ± 4.0 years old, range 26–40) were recruited from the Hokkaido University Hospital Infertility Clinic. All of the RM women had a history of two or more miscarriages (3.3 ± 2.9, range 2–15) without experiencing a live birth. Eleven women had three or more miscarriages, while nine had two miscarriages. All had been subjected to examination by ultrasound and hysterosalpingography in order to detect anatomical abnormalities of the genital tract and cervical incompetence. Measurements were made of serum testosterone, estradiol, early follicular phase FSH, LH and mid-luteal phase progesterone, and endometrial biopsy was performed. Blood analyses were carried out for syphilis, anti-nuclear antibody (ANA), anti-DNA antibody, lupus anticoagulant, anti-cardiolipin antibody (aCL), {beta}2-GPI-dependent aCL, and haemostatic molecular markers that included activated partial thromboplastin time, protein C activity, d-dimer and antithrombin III. Karyotyping and screening of infectious agents in all couples were also performed.

During the study period, of 41 consecutive RM women, nine had endocrine abnormalities, four had haematological abnormalities, three had uterine anomaly or myoma, three had antiphospholipid antibody syndrome or definite autoimmune disease, and two had chromosome translocation. The remaining 20 women without plausible causes were classified as having unexplained aetiology (48.8%). Among these 20 women with unexplained aetiology, only two showed a positive test for ANA without any definite diagnosis of autoimmune disease or the presence of antiphospholipid antibody. The other 18 women carried no autoantibody.

The control group consisted of 17 non-pregnant women (34.9 ± 6.0 years old, range 26–44) who had experienced one or more normal live births without a history of miscarriage or ectopic pregnancy. All control women had regular menstrual periods.

Endometrial samples were obtained from the RM and control women by using an endometrial cell sampler, ENDOCELL (WALLACH Surgical Devices, Inc., Orange, CT), with informed consent during the mid-luteal phases of their menstrual cycle. The mid-luteal phases (5th–9th day of a high phase) in RM women were confirmed by basal body temperature.

This study has been approved by the ethics committee of the Graduate School of Medicine, Hokkaido University.

Flow cytometric analysis
The endometria were suspended in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and 0.1% sodium azide, and were minced by surgical scissors and strained through a nylon mesh (59 µm). A lysing solution containing NH4Cl and EDTA was added for 10 min at room temperature to lyse the erythrocytes. The cells were washed twice with PBS and resuspended with 1 ml of PBS before flow cytometric analysis. For the NK cell analyses, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (Becton Dickinson & Co., San Jose, CA) and phycoerythrin (PE)-conjugated anti-CD56 (Beckman Coulter, Inc., Fullerton, CA). For the NKT cell analyses, the cells were stained with phycoerythrin-cyanine 5 (PC5)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD4 and anti-CD8, PE-conjugated anti-V{alpha}24 and FITC-conjugated anti-V{beta}11 monoclonal antibodies (mAbs), all purchased from Immunotech. Two- and four-colour flow cytometric analyses were carried out using FACS Calibur flow cytometry (Becton Dickinson & Co.) and CellQuest Software (Figure 1).



View larger version (62K):
[in this window]
[in a new window]
 
Figure 1. The frequencies of CD3+CD4CD8 V{alpha}24+V{beta}11+ NKT cells in endometrium. Endometrial cells were stained with CD3, CD4, CD8, V{alpha}24 and V{beta}11 monoclonal antibody, as described in Materials and methods. A gate was set on lymphocytes by characteristic forward scatter (FSC) and side scatter (SSC) (R1). The analyses gate was set for CD3+CD4 CD8 lymphocytes (R4) and they were identified further by PE (V{alpha}24) and FITC (V{beta}11) fluorescence. The CD3+CD4CD8V{alpha}24+V{beta}11+ NKT cell population was detected as distinct dots in the upper right quadrant.

 
The intracellular cytokines were stained according to the method of Picker et al. (1995Go). The cells in the endometrium were stimulated with phorbol myristate acetate (PMA) (25 ng/ml) and ionomycin (2 µg/ml) in the presence of brefeldin A (10 µg/ml) for 4 h. These cells were stained with PC5-conjugated anti-CD4 mAb (Immunotech). The cells were washed and fixed with fixation buffer (Becton Dickinson & Co.), and then washed with permeabilization buffer (Becton Dickinson & Co.). These fixed and permeabilized cells were stained with PE-labelled anti-IL-4, FITC-labelled anti-IFN-{gamma} or anti-TNF-{alpha} mAb (Becton Dickinson & Co.). A three-colour flow cytometric analysis for CD4, IL-4 and IFN-{gamma} and a two-colour flow cytometric analysis for CD4 and TNF-{alpha} were performed. The Th1 cells were defined as CD4+ lymphocytes with intracellular IFN-{gamma} but without intracellular IL-4. The Th2 cells were defined as CD4+ lymphocytes with intracellular IL-4 but without intracellular IFN-{gamma}. The Mann–Whitney U-test was used to analyse the results for unpaired data. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
There was no difference in the percentages of lymphocytes and CD3+ cells in the endometrium between the two groups (Table I). However, the percentages of CD3+ cells in lymphocytes were significantly lower in RM women than in control women (mean ± SD; 40.3 ± 13.3 versus 56.5 ± 13.5%).


View this table:
[in this window]
[in a new window]
 
Table I. CD3+ lymphocyte, natural killer cell and natural killer T cell population in the endometrium
 
The percentages of NK cells including CD56+ cells, CD56+CD16+ cells and CD56+CD16 cells in lymphocytes, or the CD16+ cell percentage in CD56+ cells showed no statistically significant difference between the two groups (Table I).

NKT cell percentages in V{alpha}24+V{beta}11+ cells/CD3+CD4CD8 cells, V{alpha}24+ cells/CD3+CD4CD8 cells, V{alpha}24+V{beta}11+ CD3+CD4CD8 cells/lymphocytes or V{alpha}24+CD3+CD4CD8 cells/lymphocytes showed no difference between the two groups (Table I).

Table II shows IFN-{gamma}-, TNF-{alpha}- and IL-4-expressing cell populations and ratios in CD4+ cells. In RM women compared with controls, CD4+IFN-{gamma}+ cell percentages (28.4 ± 13.4% versus 39.5 ± 12.1%, respectively) and CD4+TNF-{alpha}+ cell percentages (32.9 ± 17.6% versus 45.8 ± 12.3%, respectively) were significantly lower than those in control women. CD4+IFN-{gamma}IL-4 cell percentages in RM women were significantly higher than those in controls (71.0 ± 13.6 versus 59.6 ± 11.7%). The CD4+IL-4+/CD4+IFN-{gamma}+ (Th2 + Th0/Th1 + Th0) cell ratio, CD4+IL-4+IFN-{gamma}/CD4+IFN-{gamma}+IL-4 (Th2/Th1) cell ratio and the CD4+IL-4+/CD4+TNF-{alpha}+ cell ratio were not different between the two groups.


View this table:
[in this window]
[in a new window]
 
Table II. IFN-{gamma}-, TNF-{alpha}- and IL-4-expressing CD4+ cell population in the endometrium
 
When RM women with a history of three or more miscarriages (n = 11) were analysed separately, their percentages of CD3+ cells in lymphocytes (40.1 ± 11.9%), CD4+IFN-{gamma}+ cells (27.8 ± 13.2%) and CD4+TNF-{alpha}+ cells (28.3 ± 17.3%) were significantly lower; and CD4+IFN-{gamma}IL-4 cells (71.5 ± 13.3%) were significantly higher than those in controls (Figure 2). Thus, the significant differences in these parameters were demonstrated in women with recurrent miscarriage (n = 11) as well as women with two or more miscarriages (n = 20).






View larger version (44K):
[in this window]
[in a new window]
 
Figure 2. Distribution of individual measurements of the lymphocyte subpopulations. (A) CD3+ cells/lymphocytes; (B) IFN-{gamma}+ CD4+ cells; (C) TNF-{alpha}+ CD4+ cells; and (D) IFN-{gamma}IL-4 CD4+ cells. RM = repeated miscarriage. Bars indicate mean values. Closed circles = women who experienced three or more miscarriages (n = 11); open circles = women who experienced two miscarriages (n = 9); open squares = fertile controls (n = 17).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
In the present study, abnormalities of the NK cell population in the endometrium were not detected in RM women. Another investigation also reported that there was no difference in NK cell numbers in the endometrium (during the peri-implantation period) between RM women and controls, and that the NK cell population in the endometrium fails to predict subsequent pregnancy outcome (Michimata et al., 2002Go). The endometrial NK cell percentages in the non-pregnant status might not be related to the pathophysiology of RM.

Recently, CD1d antigen has been found to be expressed on trophoblasts and to interact with NKT cells in the decidua. The decidual NKT cells produce much more granulocyte–macrophage colony-stimulating factor (GM-CSF) and IFN-{gamma} than do peripheral NKT cells (Boyson et al., 2002Go). Murine studies have demonstrated that fetal growth and viability are jeopardized in the absence of maternal GM-CSF (Robertson et al., 1999Go) and that IFN-{gamma} plays a crucial role in the murine implantation phenomenon (Ashkar and Croy, 1999Go; Ashkar et al., 2000Go). Therefore, NKT cells seem to be beneficial to maintaining the normal reproduction process. In contrast, it has been demonstrated in a murine study that NKT cells can provoke fetal resorption if overstimulated by a specific ligand for V{alpha}14+NKT cells, {alpha}-galactosylceramide ({alpha}GalCer) (Ito et al., 2000Go). In the previous study, we found no difference in V{alpha}24+V{beta}11+ NKT cell percentages among CD3+CD4CD8 cells in peripheral blood between RM women (mean ± SD; 0.61 ± 0.51%) and fertile controls (0.46 ± 0.47%). In the present study, we assessed the NKT cell population in the endometrium and also found no difference in the V{alpha}24+V{beta}11+ NKT cell population between RM women (1.90 ± 1.78%) and fertile control women (2.34 ± 1.76%). However, NKT cell accumulation in the endometrium compared with that in peripheral blood was noted; this phenomenon was in agreement with the results of another study (Tsuda et al., 2001Go). Although NKT cells were not found to be involved in RM aetiologies, these cells might play some roles in the reproduction process.

In the present study, we demonstrated that the percentage of CD3+ cells in endometrial lymphocytes was decreased in comparison with controls, while the percentage of IFN-{gamma}IL-4 cells in CD4+ cells was increased. The percentages of IFN-{gamma}+ cells and TNF-{alpha}+ cells in CD4+ cells were also decreased in RM women. These findings suggest the diminution of T-cell activity rather than accentuation in the endometrium of RM women. One study has demonstrated lower IFN-{gamma} expression in the peripheral CD4+ and CD8+ lymphocytes of RM women than in those of controls (Rein et al., 2002Go). We have also found Th2 and Tc2 predominance of peripheral lymphocytes in non-pregnant RM women compared with fertile women (Shimada et al., 2003Go). The IFN-{gamma}+/IL-4+cell ratio and TNF-{alpha}+/IL-4+cell ratio in CD4+ and CD8+ peripheral lymphocytes have been observed to be decreased in RM women (Shimada et al. 2003Go). Thus, we confirmed the diminution of IFN-{gamma}+ and TNF-{alpha}+ cells in peripheral lymphocytes as well as in the endometrium in RM.

Murine studies have demonstrated that the predominant Th1 immunity is related to implantation failure and fetal resorptions (Krishnan et al., 1996Go). The Th2 cytokines produced at the materno-fetal interface have been thought to be beneficial to the maintenance of pregnancy because these suppress cellular cytotoxicity (Lin et al., 1993Go; Wegmann et al., 1993Go). However, a recent murine study has demonstrated that Th2 cytokines, IL-4 and IL-10, were not crucial to the successful completion of allogeneic pregnancies (Svensson et al., 2001Go). In humans, no differences in the Th2 cell marker in the endometrium during the peri-implantation period have been observed between RM women and controls, and this marker has also failed to predict subsequent pregnancy outcome (Michimata et al., 2002Go). One recent study has demonstrated that the IFN-{gamma} concentration in the uterine cavity fluid is decreased in women with recurent implantation failure (Inagaki et al., 2003Go). Another study has found decreases in the percentages of the Th1-inducing cytokine IL-12 in lymphocytes, monocytes and granulocytes derived from peripheral blood and the decidua of RM women when compared with those of normal pregnant women (Zenclussen et al., 2002Go). Using stimulated peripheral lymphocytes, it has been found that TNF-{alpha} production levels at 6–10 weeks of gestation in RM women with subsequent miscarriages are lower than those in RM women with successful reproductive outcomes (Bates et al., 2002Go). There are still many controversies concerning peripheral Th1/Th2 balance as the underlying pathophysiology and predictive values for miscarriage in RM women.

TNF-{alpha} is a primarily pro-inflammatory cytokine produced preferentially, but not uniquely, by Th1 cells like GM-CSF, and the main source of this cytokine is assumed to be macrophages/monocytes (Cherwinski et al., 1987Go). Embryo-protective effects of TNF-{alpha} against teratogenic stress have been discovered recently (Torchinsky et al., 2003Go). IFN-{gamma} has been known to contribute essentially to implantation, decidual integrity and placental growth (Ashkar et al., 2000Go); therefore, decreased production of TNF-{alpha} and IFN-{gamma} at the materno-fetal interface could adversely affect implantation and fetal development. Our results in the present and previous studies in addition to the recent findings of others suggest that the diminution of cytokine production early in gestation rather than the Th1 dominance is related to the aetiology of RM and poor reproductive prognosis. The insemination and the implantation normally induce remarkable changes of Th cytokine expression in the endometrium and decidua (Krasnow et al., 1996Go). It is hypothesized that diminution of Th1 cytokine expression in the endometrium disturbs the induction of immune tolerance during the processes of insemination and implantation and during subsequent embryonic development.

In conclusion, we found diminution of CD3+ cells, CD4+IFN-{gamma}+ cells and CD4+TNF-{alpha}+ cells but not Th1 predominance, NK cell or NKT cell accentuation in the endometrium of RM women. The immunodystrophism, i.e. reduced Th1 cell function, in the endometrium may be the underlying pathophysiology of RM.

However, the number of subjects in this study population was rather small. The differences of cytokine expression in the endometrium between RM women and controls were not sufficient to conclude that these changes would be essential to cause RM. Further immunological characterization of T cells in endometrium as well as in the decidua would promote our understanding of the underlying pathology of RM. Further studies should be performed in order to determine the cause–effect relationship, especially in miscarriage with normal fetal chromosome karyotype.


    Acknowledgments
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
We are very grateful to Dr Eiji Nomura for his assistance in collecting control samples. This work was supported in part by a Grant-in-Aid (No. 14370521) from the Ministry of Education, Science, Sports and Culture of Japan and by a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgments
 References
 
Ashkar AA and Croy BA (1999) Interferon-gamma contributes to the normalcy of murine pregnancy. Biol Reprod 61,493–502[Abstract/Free Full Text]

Ashkar AA, Di Santo JP and Croy BA (2000) Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med 192,259–270.[Abstract/Free Full Text]

Bates MD, Quenby S Takakuwa K, Johnson PM and Vince GS (2002) Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss? Hum Reprod 17,2439–2444.[Abstract/Free Full Text]

Boyson JE, Rybalov B, Koopman LA, Exley M, Balk SP, Racke FK, Schatz F, Masch R, Wilson SB and Strominger JL (2002) CD1d and invariant NKT cells at the human maternal–fetal interface. Proc Natl Acad Sci USA 99,13741–13746.[Abstract/Free Full Text]

Cherwinski HM, Schumacher JH, Brown KD and Mosmann TR (1987) Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J Exp Med 166,1229–1244.[Abstract]

Dang Y and Heyborne KD (2001) Cutting edge: regulation of uterine NKT cells by a fetal class I molecule other than CD1. J Immunol 166,3641–3644.[Abstract/Free Full Text]

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

Inagaki N, Stern C, McBain J, Lopata A, Kornman L and Wilkinson D (2003) Analysis of intra-uterine cytokine concentration and matrix-metalloproteinase activity in women with recurrent failed embryo transfer. Hum Reprod 18,608–615.[Abstract/Free Full Text]

Ito K, Karasawa M, Kawano T, Akasaka T, Koseki H, Akutsu Y, Kondo E, Sekiya S, Sekikawa K, Harada M et al. (2000) Involvement of decidual V{alpha}14 NKT cells in abortion. Proc Natl Acad Sci USA 97,740–744.[Abstract/Free Full Text]

Joyce S (2001) CD1d and natural T cells: how their properties jump-start the immune system. Cell Mol Life Sci 58,442–469.[Medline]

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

Krishnan L, Guilbert LJ, Wegmann TG, Belosevic M and Mosmann TR (1996) T helper 1 response against Leishmania major in pregnant C57BL/6 mice increases implantation failure and fetal resorptions. Correlation with increased IFN-gamma and TNF and reduced IL-10 production by placental cells. J Immunol 156,653–662.[Abstract]

Kwak-Kim JY, Chung-Bang HS, Ng SC, Ntrivalas EI, Mangubat CP, Beaman KD, Beer AE and Gilman-Sachs A (2003) Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF. Hum Reprod 18,767–773.[Abstract/Free Full Text]

Lin H, Mosmann TR, Guilbert L, Tuntipopipat S and Wegmann TG (1993) Synthesis of T helper 2-type cytokines at the maternal–fetal interface. J Immunol 151,4562–4573.[Abstract/Free Full Text]

Michimata T, Ogasawara MS, Tsuda H, Suzumori K, Aoki K, Sakai M, Fujimura M, Nagata K, Nakamura M and Saito S (2002) Distributions of endometrial NK cells, B cells, T cells, and Th2/Tc2 cells fail to predict pregnancy outcome following recurrent abortion. Am J Reprod Immunol 47,196–202.[CrossRef][Medline]

Mosmann TR and Coffman RL (1989) Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7,145–173.[CrossRef][Medline]

Picker LJ, Singh MK, Zdraveski Z, Treer JR, Waldrop SL, Bergstresser PR and Maino VC (1995) Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86,1408–1419.[Abstract/Free Full Text]

Raghupathy R, Makhseed 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.[CrossRef][Medline]

Rein DT, Schondorf T, Gohring UJ, Kurbacher CM, Pinto I, Breidenbach M, Mallmann P, Kolhagen H and Engel H (2002) Cytokine expression in peripheral blood lymphocytes indicates a switch to T (HELPER) cells in patients with preeclampsia. J Reprod Immunol 54,133–142.[CrossRef][Medline]

Robertson SA, Roberts CT, Farr KL, Dunn AR and Seamark RF (1999) Fertility impairment in granulocyte–macrophage colony-stimulating factor-deficient mice. Biol Reprod 60,251–261.[Abstract/Free Full Text]

Shimada S, Iwabuchi K, Hirayama EK, Morikawa M, Sakuragi N, Onoé K, Minakami H and Yamada H (2003) No difference in natural-killer-T cell population, but Th2/Tc2 predominance in peripheral blood of recurrent aborters. Am J Reprod Immunol 50,334–339.[CrossRef][Medline]

Svensson L, Arvola M, Sallstrom MA, Holmdahl R and Mattsson R (2001) The Th2 cytokines IL-4 and IL-10 are not crucial for the completion of allogeneic pregnancy in mice. J Reprod Immunol 51,3–7.[CrossRef][Medline]

Torchinsky A, Shepshelovich J, Orenstein H, Zaslavsky Z, Savion S, Carp H, Fain A and Toder V (2003) TNF-alpha protects embryos exposed to developmental toxicants. Am J Reprod Immunol 49,159–168.[CrossRef][Medline]

Tsuda H, Sakai M, Michimata T, Tanebe K, Hayakawa S and Saito S (2001) Characterization of NKT cells in human peripheral blood and decidual lymphocytes. Am J Reprod Immunol 45,295–302.[CrossRef][Medline]

Wegmann TG, Lin H, Guilbert L and Mosmann TR (1993) Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 14,353–356.[CrossRef][Medline]

Yamada H, Kato EH, Kobashi G, Ebina Y, Shimada S, Morikawa M, Sakuragi N and Fujimoto S (2001) High NK cell activity in early pregnancy correlates with subsequent abortion with normal chromosomes in women with recurrent abortion. Am J Reprod Immunol 46,132–136.[CrossRef][Medline]

Yamada H, Morikawa M, Kato EH, Shimada S, Kobashi G and Minakami H (2003) Preconceptional natural-killer-cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. Am J Reprod Immunol 50,351–354.[CrossRef][Medline]

Zenclussen AC, Fest S, Busse P, Joachim R, Klapp BF and Arck PC (2002) Questioning the Th1/Th2 paradigm in reproduction: peripheral levels of IL-12 are down-regulated in miscarriage patients. Am J Reprod Immunol 48,245–251.[CrossRef][Medline]

Submitted on July 28, 2003; accepted on January 5, 2004.