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

Human Reproduction Decrease of T-helper 2 and T-cytotoxic 2 cells at implantation sites occurs in unexplained recurrent spontaneous abortion with normal chromosomal content T.Michimata 2 M.Sakai 2 S.Miyazaki 2 M.S.Ogasawara 1 K.Suzumori 1 K.Aoki 0 K.Nagata 3 S.Saito 2 0 Department of Obstetrics and Gynecology, Johsai Hospital , Nagoya-shi, Aichi, 453-0815 1 Department of Obstetrics and Gynecology, Nagoya City University , Nagoya-shi, Aichi, 467-8601 2 Department of Obstetrics and Gynecology, Toyama Medical and Pharmaceutical University , 2630 Sugitani , Toyama-shi, Toyama , 930-0194 3 Research and Development Center , BML, Kawagoe-shi, Saitama, 350-1101 , Japan 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 classied 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 parafn-embedded decidual sections for a specic 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. CRTH2/decidua implantation/recurrent spontaneous abortion/Th2 Introduction 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, 1953; Clark et al., 1999). 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)-g, and tumour necrosis factor (TNF)-b, and the up-regulation of Th2 and Tc2 cells, which produce IL-4, IL-6, IL-10 and IL-13 (Lin et al., 1993; Wegmann et al., 1993; Piccinni and Romagnani, 1999; Saito, 2000). 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., 1993; Lim et al., 1998; Piccinni et al., 1998; Saito et al., 1999; Tsuda et al., 2001; 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., 1995; Krishnan et al., 1996). There is evidence of a Th1-type response associated with recurrent spontaneous abortion (RSA) (Hill et al., 1995; Shaarawy and Nagui, 1997; Vives et al., 1999; Hayakawa et al., 2000; Jenkins et al., 2000; Lim et al., 2000; Makhseed et al., 2000; 2001; Raghupathy et al., 2000). Piccinni et al. (1998) 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., 1980; Stern et al., 1996). 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 immunouorescence staining of a chemo-attractant receptor-homologous molecule (CRTH2) (Michimata et al., 2002a). Here, we studied the localization of CRTH2+ Th2 cells Graviditya Normal controls (n = 15) 31.0 6 5.49 3.27 6 0.59 Nonec Women with recurrent abortion 30.7 6 3.72 3.64 6 1.21 31.3 6 2.95 5.40 6 1.71 Values are shown as means 6 SD. aAbortion times and live birth times. bIncludes the abortions of this study. cControl group had no history of spontaneous abortion. RSA-A = unexplained recurrent spontaneous abortion with abnormal chromosomal content; RSA-N = unexplained recurrent spontaneous abortion with normal chromosomal content. and CRTH2+ Tc2 cells in the decidua in RSA of normal embryo and abnormal embryos. Materials and methods 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, b2-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 identied 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 38) and 3.6 (range 36) respectively. Specimen Human decidual tissues (610 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. 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; t (...truncated)