Decrease of T-helper 2 and T-cytotoxic 2 cells at implantation sites occurs in unexplained recurrent spontaneous abortion with normal chromosomal content

T. Michimata1, M. Sakai1, S. Miyazaki1, M.S. Ogasawara2, K. Suzumori2, K. Aoki3, K. Nagata4 and S. Saito1,5

1 Department of Obstetrics and Gynecology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama-shi, Toyama, 930-0194, 2 Department of Obstetrics and Gynecology, Nagoya City University, Nagoya-shi, Aichi, 467-8601, 3 Department of Obstetrics and Gynecology, Johsai Hospital, Nagoya-shi, Aichi, 453-0815 and 4 Research and Development Center, BML, Kawagoe-shi, Saitama, 350-1101, Japan

5 To whom correspondence should be addressed. e-mail: s30saito{at}ms.toyama-mpu.ac.jp


    Abstract
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 Abstract
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 Materials and methods
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BACKGROUND: In normal pregnancy, predominant type 2 cytokines help maintain pregnancy, and a T-helper (Th)1 type response is associated with unexplained recurrent spontaneous abortion (RSA). However, Th2 and T-cytotoxic (Tc)2 cells have not been localized at the implantation site in RSA. METHODS: Twenty-one cases with RSA were classified into RSA with normal chromosomal content (RSA-N, n = 10) and RSA with abnormal chromosomal content (RSA-A, n = 11). As a control, we selected 15 gestational age-matched cases of induced abortion with no history of spontaneous abortion. We immunostained paraffin-embedded decidual sections for a specific Th2 and Tc2 cell marker termed ‘chemo-attractant receptor-homologous molecule expressed on Th2 cells (CRTH2)’ and T-cell markers CD3 and CD8. The numbers and percentages of Th2 (CRTH2+CD8CD3+) and Tc2 (CRTH2+CD8+) cells were compared between the decidua basalis and decidua parietalis. RESULTS: Th2 and Tc2 cells accumulated in the decidua basalis in normal pregnancy. Accumulation of Tc2 cells and both Th2 and Tc2 cells decreased in the decidua basalis in RSA-A and RSA-N respectively. The number and percentage of Th2, and Tc2 cells in the decidua parietalis were similar in normal pregnancy, RSA-A and RSA-N. CONCLUSION: Decreased Th2 and Tc2 cells at the implantation site may contribute to RSA-N.

Key words: CRTH2/decidua implantation/recurrent spontaneous abortion/Th2


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Successful human pregnancy appears to be an immunological paradox, in that the fetus represents a semi-allograft developing in the potentially hostile environment of the maternal immune system (Medawar, 1953Go; Clark et al., 1999Go). One important mechanism involves the down-regulation of the cellular immune response, which has been shown to be dependent upon the suppression of T-helper (Th) 1 and T-cytotoxic (Tc) 1 cells, which produce interleukin (IL)-2, interferon (IFN)-{gamma}, and tumour necrosis factor (TNF)-{beta}, and the up-regulation of Th2 and Tc2 cells, which produce IL-4, IL-6, IL-10 and IL-13 (Lin et al., 1993Go; Wegmann et al., 1993Go; Piccinni and Romagnani, 1999Go; Saito, 2000Go). Previous investigations of Th1/Th2 immune responses during pregnancy were able to show that a distinct shift towards Th2-type reactions occurs, especially at the feto-maternal interface (Lin et al., 1993Go; Lim et al., 1998Go; Piccinni et al., 1998Go; Saito et al., 1999Go; Tsuda et al., 2001Go; 2002).

Cases of pregnancy failure, such as spontaneous abortion, have been studied for altered immune responses and changes in cytokine patterns in mice (Chaouat et al., 1995Go; Krishnan et al., 1996Go). There is evidence of a Th1-type response associated with recurrent spontaneous abortion (RSA) (Hill et al., 1995Go; Shaarawy and Nagui, 1997Go; Vives et al., 1999Go; Hayakawa et al., 2000Go; Jenkins et al., 2000Go; Lim et al., 2000Go; Makhseed et al., 2000Go; 2001; Raghupathy et al., 2000Go). Piccinni et al. (1998Go) found that IL-4, IL-10 and leukaemia inhibitory factor (LIF) production by decidual T cells is decreased in women with RSA, suggesting a potential functional link between a Th2-type cytokine bias and successful continuation of pregnancy. In humans, 50% of unexplained RSA may be attributed to chromosome abnormalities in the embryonic trophoblast (Simpson., 1980Go; Stern et al., 1996Go). The immunological environment, i.e. the Th1/Th2 balance, might be disturbed in RSA of normal embryos. However, localization of Th2 and Tc2 cells at the implantation site in RSA has not been reported. Recently, we developed a novel method to detect Th2 and Tc2 cells by immunofluorescence staining of a chemo-attractant receptor-homologous molecule (CRTH2) (Michimata et al., 2002aGo). Here, we studied the localization of CRTH2+ Th2 cells and CRTH2+ Tc2 cells in the decidua in RSA of normal embryo and abnormal embryos.


    Materials and methods
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 Materials and methods
 Results
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Women with recurrent abortion
This study group comprised 21 women with three or more unexplained RSA who have no children. They were resistered at the subspecial clinic for such patients at Nagoya City University Hospital, Japan. All patients had completed the clinic’s diagnostic protocol, including chromosomal analysis of both partners, hysterosalpingography, pelvic ultrasonography, endometrial biopsy timed according to LH determinations and endometrial dating by histological examination, mid-luteal phase progesterone and estradiol measurements, screening for lupus anticoagulant, {beta}2-glycoprotein-dependent anticardiolipin antibodies, and blood tests to detect hyperthyroidism, diabetes mellitus, hyperprolactinaemia, and infections, such as Chlamydia spp. No subjects had a cause of recurrent abortion identified by this protocol. Chorionic villi were sampled from these patients for cytogenic analysis. Karyotypes determined from the examination of chorionic villi were normal in 10 cases (RSA-N) and abnormal in 11 cases (RSA-A) (monosomy one case; trisomy nine cases; tetraploid one case). The mean number of pregnancy loss of RSA-N and RSA-A is 5.4 (range 3–8) and 3.6 (range 3–6) respectively.

Specimen
Human decidual tissues (6–10 weeks gestation) were obtained from induced abortions (n = 15) and RSA cases (n = 21) (Table I). RSA cases were admitted to Nagoya City University Hospital for rest for ~1 month at 4 weeks gestation to avoid possible external risk factors. Gestational age was calculated from basal body temperature charts. Ultrasonography was performed twice per week during pregnancy. When spontaneous abortion was diagnosed, dilatation and curettage were performed.


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Table I. Comparison of clinical data from patients with unexplained recurrent abortions and normal controls
 
Women with gravidity at Toyama Medical and Pharmaceutical University Hospital were recruited over the same period. Fifteen cases were selected as control subjects. These subjects had no history of spontaneous abortion. Gestational ages were calculated from last menstrual period. Dilatation and curettage was performed in 15 cases; the karyotype of each aborted conceptus was determined. All 15 cases had normal karyotypes of aborted conceptus. Informed consent was obtained from all patients. To avoid maternal blood contamination, decidual tissues were washed in phosphate-buffered saline (PBS) twice and then fixed in 10% neutral-buffered formalin for 48 h and embedded in paraffin.

Immunofluorescence staining
Decidual T cells were stained for CRTH2, in addition to CD3 and CD8. Briefly, after deparaffinization, antigen retrieval was performed by autoclaving at 120°C for 10 min. Sections were washed in PBS and incubated for 10 min in 10% normal goat serum prior to application of primary antibodies. For double staining, sections were incubated overnight at 4°C with a combination of rat monoclonal anti-human CRTH2 antibody (5 µg/ml) which we established (Nagata et al., 1999Go) and mouse monoclonal anti-human CD3 antibody (5 µg/ml; Novocastra, UK) or anti-human CD8 antibody (5 µg/ml; Dako Japan, Tokyo). Sections were washed in PBS and incubated for 30 min with Alexa Fluor 594-labelled goat anti-rat IgG (5 µg/ml; Molecular Probes, USA) and Alexa Fluor 488-labelled goat anti-mouse IgG (5 µg/ml; Molecular Probes). Sections were mounted with SlowFade antifade kits (Molecular Probes) and examined under a confocal laser scanning microscope (LSM510; Carl Zeiss, Japan). All images were processed with LSM510 version 2.02 software for image analysis.

Since commercially available CD4 monoclonal antibody was not effective in staining paraffin-embedded tissue samples, the numbers of CD4+ T cells were calculated by subtracting the CD8+ cell count from the CD3+ count, as reported previously (Michimata et al., 2002aGo).

Quantification and data analysis
We stained serial paraffin-embedded sections immunohistochemically using anti-cytokeratin antibodies. The implantation sites were identified by cytokeratin staining to detect trophoblasts and epithelial cells in the decidua, and the decidual tissues were classified as decidua basalis (presence of invasive trophoblast and gland), or decidua parietalis (no invasive trophoblast or gland present). The number of CRTH2+CD3+, CRTH2+CD8+, CRTH2+CD3+CD8 cells per five unique fields were counted under a confocal laser scanning microscope, and the ratios for CRTH2+CD3+/CD3+, CRTH2+CD8+/CD8+ and CRTH2+CD3+CD8/CD3+CD8 cells were calculated by one physician of Toyama Medical and Pharmaceutical University. The information of karyotype of the fetus was not revealed to Toyama Medical and Pharmaceutical University staff.

As reported previously, since CRTH2+CD3cells are not detected by this method, we defined CRTH2+CD8+ cells as Tc2 cells and CRTH2+CD3+CD8 cells as Th2 cells (Michimata et al., 2002aGo).

Statistical analysis
Values are expressed as the mean ± SD. Statistical differences between groups were determined by two-sided t-test or one-way ANOVA, followed by Fisher’s protected least significant difference test (Hsu, 1996Go). P < 0.05 was accepted as significant.


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Numbers of Th2 and Tc2 cells in the decidua basalis and decidua parietalis
CRTH2+CD3+T (type 2) cells, CRTH2+CD3+CD8 T (Th2) cells and CRTH2+CD8+ T (Tc2) cells were observed around decidual vessels, endometrial gland cells and extravillous trophoblasts.

The numbers of type 2 cells, Tc2 cells and Th2 cells in the decidua basalis in subjects with a normal pregnancy, RSA-A and RSA-N were significantly higher than those in the decidua parietalis (Table II and Figure 1). However, the numbers of type 2 T cells, Th2 and Tc2 cells in the decidua basalis of RSA-A and RSA-N were significantly lower than those of normal pregnancy subjects (Table II and Figure 1).


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Table II. Numbers of type 2, Tc2 and Th2 T cells in the decidua
 


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Figure 1. The numbers and the percentages of type 2 (CRTH2+CD3+), Tc2 (CRTH2+CD8+) and (CRTH2+CD4+) T cells in the decidua basalis (open bars) and decidua parietalis (closed bars) in normal pregnancy, RSA-A and RSA-N. Values are shown as means ± SD and statistical differences were calculated by one way ANOVA. *P < 0.01, **P < 0.025, ***P < 0.05.

 
There was a difference in miscarriage history in RSA-A (majority with three abortions) and RSA-N (majority with more than four abortions) (Table I). However, the numbers of type 2, Th2 and Tc2 cells at decidua basalis and decidua parietalis were not significantly different between RSA cases with three abortions and RSA cases with more than four abortions.

Interestingly, no significant differences were noted in the number of type 2 cells, Th2 and Tc2 cells in the decidua parietalis between normal pregnancy, RSA-A and RSA-N (Figure 1).

Ratios of type 2/CD3+ T cells, Th2/CD4+ T cells and Tc2/CD8+ T cells in the decidua basalis and decidua parietalis
Since the numbers of CD3+ T, CD4+ T and CD8+ T cells at the decidua basalis were different in normal pregnancy, RSA-N and RSA-A, the type 2 immunoenvironment is best characterized by studying the ratios of type 2/CD3+, Th2/CD4+ T and Tc2/CD8+ T cells.

The ratios of type 2/CD3+, Tc2/CD8+ and Th2/CD4+ cells in the decidua basalis of normal pregnancy were significantly higher than those in the decidua parietalis, indicating accumulation of type 2, Tc2 and Th2 cells at the implantation site (Table III and Figure 1).


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Table III. Percentages of type 2, Tc2 and Th2 T cells in decidua
 
The type 2/CD3+ cell ratio in the decidua basalis of RSA-A was significantly higher than that in the decidua parietalis (Table III and Figure 1).

The type 2/CD3+ cell ratio in the decidua basalis was similar in normal pregnancy and RSA-A, suggesing that type 2 cells accumulated in RSA-A. On the other hand, no significant difference was noted in the type 2/CD3+ cell ratio between the decidua basalis and decidua parietalis in RSA-N, suggesting that lowering accumulation of type 2 cells occurred in RSA-N. Moreover, the type 2/CD3+ cell ratio in the decidua basalis in RSA-N was significantly lower than in normal pregnancy.

Next, we checked the distribution of Tc2 cells and Th2 cells in the decidua. The ratio of Tc2/CD8+ T cells in decidua basalis in normal pregnancy was significantly higher than that in the decidua parietalis, again suggesting that Tc2 cells accumulated at the implantation site in normal pregnancy. The Tc2/CD8+ T cell ratios in these locations were similar in RSA-A and RSA-N, suggesting that lowering accumulation of Tc2 cells occurred in the decidua basalis in RSA-A and RSA-N (Table III and Figure 1).

The ratio of Th2 cells/CD4+ T cells in the decidua basalis of normal pregnancy was significantly higher than that in the decidua parietalis. The result was similar in RSA-A, suggesting that accumulation of Th2 cells occurred not only in normal pregnancy, but also in RSA-A. However, this ratio was also similar in these locations in RSA-N. The Th2 cells/CD4+ ratio in the decidua basalis in RSA-N was significantly lower than that in normal pregnancy or RSA-A, suggesting that lowering accumulation of Th2 cells did occur (Table III and Figure 1).

The ratios of type 2 cells, Th2 cells and Tc2 cells at decidua basalis and decidua parietalis were not different between RSA cases with three abortions and RSA with more than four abortions.

The ratios of type 2/CD3+, Th2 /CD4+ and Tc2 /CD8+cells in the decidua parietalis were similar in normal pregnancy, RSA-A and RSA-N.

In summary, both Th2 and Tc2 cells accumulated at the decidua basalis in normal pregnancy. Th2 cells accumulated in RSA-A but Tc2 cells did not. On the other hand, lowering accumulation of Th2 and Tc2 cells at the implantation occurred in RSA-N.


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 References
 
The aetiology of RSA is varied, and includes maternal or paternal chromosomal aberrations, uterine anatomic abnormalities, endocrine disorders, infections, and reproductive antiphospholipid syndrome. However, the aetiology is undetermined in 40–60% of women with recurrent abortion (Stray-Pederson and Stray-Pederson, 1984Go; Clifford et al., 1994Go). About half of the conceptuses of RSA have an abnormal karyotype (Stern et al., 1995Go), even though the risk for a spontaneous abortion in a subsequent pregnancy is increased when a normal embryonic karyotype is found in abortus material (Morton et al., 1987Go). There is increasing evidence that Th1-type immunity is associated with RSA and may play a role in reproductive failure in mice and in humans (Chaouat et al., 1995Go; Hill et al., 1995Go; Krishnan et al., 1996Go; Shaarawy and Nagui, 1997Go; Vives et al., 1999Go; Hayakawa et al., 2000Go; Jenkins et al., 2000Go; Lim et al., 2000Go; Makhseed et al., 2000Go; 2001; Raghupathy et al., 2000Go). So if immunocompetent cells can cause RSA, the immunological environment may be disturbing especially in cases of RSA-N, and might not be disturbing in cases of RSA-A, because in cases of RSA-A the major cause of abortion is fetal anomalies, and in cases of RSA-N abortion might be caused by maternal Th1-type immunity. Indeed, localization of Th2 and Tc2 cells in the decidua basalis was different in RSA-N and RSA-A in the present study. Recently, Bates et al. (2002Go) reported that Th2 type cytokines such as IL-4 and IL-10 by peripheral blood mononuclear cells during early pregnancy were significantly increased in women with RSA compared with pregnant controls, and there were no significant differences between the level of Th2 type cytokines in RSA patients who successfully completed their pregnancy or who went on to miscarry (Bates et al., 2002Go). It was reported that a Th1/Th2 ratio at the feto-maternal interface is different from that in the peripheral blood (Piccinni et al., 1998Go; Saito et al., 1999Go; Ho et al., 2001Go; Tsuda et al., 2001Go). A different cytokine balance could occur within the endometrium and decidua. Piccinni et al. (1998Go) reported a decreased production of IL-4 by the decidual CD4+ and CD8+ T cells of RSA patients. However, localization of Th2 and Tc2 cells at the implantation site in RSA has not been reported.

Here, we studied the localization of Th2 and Tc2 cells in the decidua parietalis and decidua basalis in cases of RSA-N and RSA-A and show that increases in the number of Th2 cells and in the percentage of Th2 cells at the decidua basalis were noted in cases of RSA-A, demonstrating that specific Th2 cell accumulation occurs at the implantation site in these cases. On the other hand, the percentage of Th2 cells at the implantation site in RSA-N was similar to that at the decidua parietalis and was significantly lower than in normal pregnancy or RSA-A, demonstrating the lowering accumulation for Th2 cells at the implantation site in RSA-N. We showed that the percentages of Tc2 cells at the implantation site in RSA-N and RSA-A were similar to those at the decidua parietalis suggesting that Tc2 cells do not increase at the implantation site in either RSA-N or RSA-A. However, accumulation of type 2 cells and Th2 cells does occur in RSA-A, suggesting that the dominance of type 2 immunity persists in RSA-A. Recruitment of Th2 and Tc2 cells into the endometrium may occur during endometrial decidualization during early pregnancy. We previously reported (Michimata et al., 2002aGo) and also observed in the present study that CRTH2+ T cells were seen around decidual blood vessels, endometrial gland cells and extravillous trophoblasts. Thus, it is likely that Th2 and Tc2 cells migrate into the materno-fetal interface by attraction of chemotactic factor(s) specific for Th2 and Tc2 cells. Factors such as TARC, the Th2 chemokine, and prostaglandin D2 are produced by trophoblast and endometrial gland cells at the implantation site (Michimata et al., 2002aGo; Tsuda et al., 2002Go). Inadequate production of these factors or inadequate type 2 cells migration from maternal decidual vessels might be present in RSA-N cases.

Inflammatory processes in spontaneous abortion might affect the localization of Th2 cells and Tc2 cells. These differences may be a consequence of the inflammatory reactions expected to happen in the decidual tissue from the RSA patients and not in the tissue from the controls. The inflammation induces accumulation of lymphocytes. However, many more CD3+ T, CD4+ T and CD8+ T cells were present in control cases than in RSA-A and RSA-N cases. Similar inflammation was present in RSA-A and RSA-N by examination by haematoxylin and eosin staining. However, localizations of type 2 and Th2 cells were different between RSA-N and RSA-A suggesting that inflammation did not cause lowering accumulation of Th2 and Tc2 cells at the implantation site. Our preliminary study showed that the localizations of Th2 and Tc2 cells of spontaneous abortion (not unexplained RSA) cases with normal chromosomal content and abnormal chromosomal content were similar to those in RSA-N and RSA-A respectively, suggesting that decreased Th2 and Tc2 cells at the implantation site are associated with abortion with normal chromosomal content. Gestational ages might affect the localization of Th2 and Tc2 cells, but gestational ages in RSA-N and RSA-A were also similar. Therefore the different localizations of type 2 cells and Th2 cells between RSA-A and RSA-N would not be induced by inflammation or gestational ages. A disturbed Th1/Th2 balance might induce abortion of a normal embryo. In this study, accumulation of type 2 cells and Th2 cells were observed in the decidua basalis of RSA-A and a decrease in the accumulation of these cells was observed in the decidua basalis of RSA-N. These findings suggest that the type 2-dominant immune environment is important to maintain pregnancy.

The decrease of both Th2 and Tc2 cells at the implantation site in RSA-N suggests that a type 2-dominant immune environment is important to maintain pregnancy. These findings support the hypothesis that a Th1-type response causes miscarriage clinically, as documented in mice. The present findings only showed that Th2 and Tc2 cells are localized in the decidua basalis, but the distribution of Th1 and Tc1 cells was unclear. It is necessary to determine the Th1/Th2 and Tc1/Tc2 ratios with RSA-N when an antibody suitable for identifying Th1 and Tc1 cells in paraffin-embedded sections can be obtained.

Further studies are needed to establish whether the decreased Th2 and Tc2 cell response during pregnancy is the cause or result of pregnancy failure. We only showed the accumulation failure of Th2 and Tc2 cell accumulation at the implantation site in RSA-N. Th2 and Tc2 cells might not accumulate after implantation in RSA-N since the number of endometrial Th2 and Tc2 cells in the mid-luteal phase of RSA when the conceptus has a normal karyotype resembles those of controls (Michimata et al., 2002bGo).

Further studies are needed to identify distinct spatial, temporal and hierarchical type 2 cytokine requirements in the maintenance of pregnancy in humans.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on October 18, 2002; resubmitted on January 10, 2003; accepted on March 21, 2003.