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

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(+)CD8(-)CD3(+)) 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.     

A novel surface molecule of Th2- and Tc2-type cells, CRTH2 expression on human peripheral and decidual CD4+ and CD8+ T cells during the early stage of pregnancy

It has been demonstrated that pregnancy induces the immunomodulation of cytokine responses away from the Th1 paradigm and towards the Th2 paradigm. In this study, we examined the expression of CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2) on decidual CD4+ and CD8+ T cells during the early stages of pregnancy. Examination of the cytokine profile revealed that CRTH2 was expressed on CD4+-type-2 T helper (Th2-type) and CD8+-type 2 T cytotoxic (Tc2-type) cells. The percentages of CRTH2+ cells in CD3+/CD4+ T cells and CD3+/CD8+ T cells were significantly higher in the decidua than in the peripheral blood. These results indicate the significance of Th2- and Tc2-type cells of the decidua in the maternal immune system, presumably through their production of cytokines which may contribute to the maintenance of pregnancy.     

Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions

BACKGROUND: This study compared Th1-Th2 cytokine profiles in a subgroup of recurrent aborters who had an abortion with those in a subgroup of recurrent aborters who had a successful pregnancy. METHODS: Fifty-four women with a history of at least three normal pregnancies, 24 women with a history of recurrent spontaneous abortion (RSA) followed by abortion (RSA-->A) and 39 women with a history of RSA followed by normal pregnancy (RSA-->N) were studied. Blood samples and placentas were obtained at the time of delivery or abortion; peripheral blood mononuclear cells were stimulated separately with phytohaemagglutinin and with autologous placental cells, and the secreted cytokines estimated. RESULTS: Peripheral blood mononuclear cells from the RSA-->N subgroup secreted higher concentrations of Th1-type cytokines as compared with normal pregnant women, indicating a higher Th1 bias in these women. However, women in the RSA-->N subgroup had significantly higher concentrations of Th2 cytokines as compared with women in the RSA-->A subgroup. A comparison of Th1:Th2 cytokine ratios indicated a higher Th2 bias in RSA-->N women as compared with RSA-->A women. CONCLUSIONS: We conclude that abortion-prone women who proceed to have successful pregnancy are more Th2-biased than abortion-prone women who abort, and that recurrent aborters who undergo spontaneous abortion have a stronger Th1 bias than aborters who have normal pregnancy.     

Preimplantation Factor (PIF) Predicts Subsequent Pregnancy Loss

PROBLEM : To evaluate the ability of preimplantation factor (PIF) measured in the lymphocyte/platelet binding assay (LPBA) to predict subsequent spontaneous abortion. METHOD : Serum from 57 women experiencing first trimester pregnancy losses were studied using the LPBA (46 women conceived after in vitro fertilization and embryo transfer for treatment of infertility and 11 with a history of unexplained recurrent spontaneous abortion conceived spontaneously). The assay employs a combination of heat inactivated sera with donor O+ lymphocytes and platelets, complement and an antibody against CD2. Chromosome analysis was performed on 32 of the abortuses. Results of PIF assay were compared between karyotypically normal and abnormal abortuses. RESULTS : PIF assay was negative in all 57 women at the time of abortion. Among 12 karyotypically normal abortuses only 1 woman (8%) had an initial positive PIF, 11 (92%) had negative PIF. Serial PIF assays were performed on 15 women. PIF assay became negative a minimum of two weeks prior to demonstration of intrauterine demise at a time when hCG concentrations remained elevated. A trend to subnormal was seen in women with normal when compared to those with abnormal abortus karyotype, but the numbers were too small to reach statistical significance (P = 0.09). CONCLUSION : Measurement of PIF throughout the first trimester of pregnancy predicts subsequent pregnancy loss.     

No difference in natural-killer-T cell population, but Th2/Tc2 predominance in peripheral blood of recurrent aborters

PROBLEM: The aim of this study was to assess the natural-killer-T (NKT) cell population and cytokine expression in the peripheral blood of women with a history of recurrent spontaneous abortion (RSA). METHOD OF STUDY: The percentages of CD3+ CD4- CD8- TCRValpha24+ Vbeta11+-NKT cells and cells expressing intracellular interferon (IFN)-gamma, interleukin (IL)-4, and tumor necrosis factor (TNF)-alpha either with CD4+ or CD8+ cells were measured by flow cytometry at the midluteal phases in 15 RSA women and 15 fertile control women. RESULTS: No significant differences in the NKT cell percentages were found between RSA and control women. However, in RSA women, the CD4+ IL-4+ cell and CD8+ IL-4+ cell percentages were significantly higher, and the Th1/Th2 and Tc1/Tc2 cell ratios were significantly lower, than those in the control. CONCLUSIONS: Th2/Tc2 dominance was found in the general circulation of RSA women; this finding provokes a new controversy on the Th1/Th2 balance concerning RSA etiology.     

Immunotherapy Before and During Pregnancy Improves Pregnancy Outcome in Women Who Suffer from Recurrent Abortion and Did Not Benefit from Immunotherapy Before Pregnancy

PROBLEM: The appropriate modality of immunotherapy with the husband's mononuclear cells in women with a history of recurrent abortion who aborted despite the immunotherapy performed before pregnancy was explored. METHOD OF STUDY: Nineteen patients who had suffered from recurrent abortion who had received the immunotherapy only before pregnancy and had aborted were treated with further immunotherapy performed either only before pregnancy or twice: before and during pregnancy. RESULTS: In 9 out of the 19 women who received further immunotherapy before pregnancy, 2 had healthy babies and 7 aborted again. In the remaining 10 patients who received further immunotherapy twice, before and during pregnancy, 8 had healthy babies and 2 aborted again. CONCLUSION: Our results indicate that immunotherapy performed before and during pregnancy produces a better outcome compared with that performed only before pregnancy, especially in patients who showed no benefit from the immunotherapy performed only before pregnancy.     

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

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.     

A paracrine role for chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) in mediating chemotactic activation of CRTH2+ CD4+ T helper type 2 lymphocytes

Activation of human CRTH2(+) CD4(+) T helper type 2 (Th2) cells with anti-CD3/anti-CD28 led to time-dependent production of prostaglandin D(2) (PGD(2)) which peaked at 8 hr. The production of PGD(2) was completely inhibited by cotreatment with the cyclo-oxygenase inhibitor diclofenac (10 microm) but was not affected by cotreatment with ramatroban, a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Supernatants from activated CRTH2(+) CD4(+) Th2 cells caused a concentration-dependent increase in the migration of naive CRTH2(+) CD4(+) Th2 cells compared to supernatants from unstimulated CRTH2(+) CD4(+) Th2 cells. The level of chemotactic activity peaked at 8 hr after activation, corresponding to the peak levels of PGD(2), but production of chemotactic activity was only partially inhibited by the cyclo-oxygenase inhibitor diclofenac. In contrast, ramatroban completely inhibited the chemotactic responses of naive Th2 cells to supernatants from activated CRTH2(+) CD4(+) Th2 cells collected up to 8 hr after activation, although supernatants collected 24 hr after activation were less sensitive to inhibition by ramatroban. The selective TP antagonist SQ29548 did not inhibit migration of Th2 cells, implicating CRTH2 in this response. These data suggest that CRTH2 plays an important paracrine role in mediating chemotactic activation of Th2 cells. Interestingly, although PGD(2) is produced from Th2 cells and contributes to this paracrine activation, it appears that additional CRTH2 agonist factors are also produced by activated Th2 cells and the production of these factors occurs independently of the cyclo-oxygenase pathway of the arachidonic acid metabolism.     

Circulating cytokines and CD30 in normal human pregnancy and recurrent spontaneous abortions

Concentrations of the T-helper (Th) 1 cytokines interleukin (IL)-2, tumour necrosis factor (TNF) -alpha, TNF-beta and interferon-gamma, Th2 cytokines IL-4, IL-5, IL-6, IL-10 as well as those of soluble CD30 in sera have been examined during the three trimesters of gestation, at delivery in normal pregnancy, and at the time of spontaneous abortion in women with a history of unexplained recurrent spontaneous abortion (RSA). Significantly higher concentrations of the Th2 cytokines IL-6 and IL-10 were found at normal delivery than in women with RSA, and conversely significantly increased concentrations of the Th1-type cytokine TNF-alpha were found in RSA as compared with successful pregnancy. In abortion-prone women who had a successful pregnancy, significantly higher concentrations of IL-6 and significantly lower concentrations of TNF-alpha were found as compared with abortion-prone women who had another abortion, supporting the notion that Th2- and Th1-bias are associated with successful and unsuccessful pregnancy respectively. Serum CD30 concentrations did not correlate with the outcome of pregnancy. These findings support observations drawn from experiments on the cytokine secretion profiles of peripheral blood mononuclear cells and decidual lymphocytes which suggest that normal pregnancy is Th2-biased and that unexplained RSA is associated with Th1-type reactivity.     

Association between infertility and spontaneous abortion.

Women experiencing recurrent spontaneous abortion have a higher frequency of infertility than that expected in the general population. To further define the relationships between infertility and spontaneous abortion, the obstetrical histories of 43 women with unexplained secondary infertility were evaluated for the frequency of spontaneous abortion. Of the 88 pregnancies studied, 39 (44%) resulted in spontaneous abortion. Women with unexplained secondary infertility experienced a three-fold increase (P less than 0.0001) in the frequency of spontaneous abortions and half the number of live births (P less than 0.0001) compared with the general population. We conclude that the association between infertility and spontaneous abortion includes a higher frequency of spontaneous abortion among infertile couples as well as a higher prevalence of infertility among recurrent spontaneous aborters compared with the general population.     

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease

Cells expressing the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the chemokine C receptor (CCR)4 were consistently detected in the circulation of healthy subjects, whereas numbers of cells expressing CCR3 were much lower. While all CCR4+ cells were T cells, a small proportion of CRTH2+, and about a half of the few CCR3+ cells were basophils. Only CRTH2+ T cells contained Th2 or Tc2 cells, but neither Th0 or Tc0, nor Th1 or Tc1 cells, although not all of them produced Th2-type cytokines. By contrast, CCR4+ T cells contained both Th2 or Tc2 and Th0 or Tc0 cells and even Th1 or Tc1 cells, whereas the few CCR3+ T cells were not clearly classifiable for their cytokine profile. CRTH2+ T lymphocytes were virtually devoid of chemokine CX receptor (CXCR)3+ and CCR5+ cells, but enriched in CCR3+ and CCR4+ cells. By contrast, CCR3+ or CCR4+ T cells did not show a similar clear-cut dichotomy in the expression of CCR5/CXCR3 or CCR3/ CCR4. Subjects with atopic dermatitis or HIV infection with low levels of circulating CD4+ T cells revealed a significant increase of CRTH2+ cells within both the CD4+ and the CD8+ T cell subset. These data support the concept that at present CRTH2 is the more reliable marker for detection of both human Th2 and Tc2 cells in health and disease.     

自身抗体与不孕及自发性流产关系的探讨

目的检测抗心磷脂抗体(ACA)和抗精子抗体(AsAb)两种自身抗体在不孕及自发性流产患者中存在的情况,并观察应用阿司匹林治疗ACA阳性反复流产患者的临床效果。方法应用酶联免疫吸附(ELISA)法检测150例原发或继发不孕患者(不孕组)、198例自发性流产或有胚胎停育史患者(流产组)及40例正常对照组血清中的ACA及AsAb抗体。对其中53例ACA阳性反复流产患者在孕前一个月或孕早期采用低剂量阿司匹林治疗。结果不孕组及流产组ACA总阳性率分别为48.00%和50.51%,与对照组(7.50%)相比有非常显著性差异(P<0.001);不孕组及流产组AsAb阳性率分别为31.33%和25.25%,与对照组(10.00%)比较亦有显著性差异(P<0.05)。53例经治疗患者活产婴儿48例,妊娠成功率为90.57%。结论ACA和AsAb等自身抗体是导致不孕及自发性流产的免疫学因素之一,应用低剂量阿司匹林治疗ACA阳性反复流产患者是保证其妊娠成功的有效方法。     

Expression of intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,implantation failures after IVF/ET or normal pregnancy

PROBLEM: We aimed to investigate absolute counts of intracellular T helper 1 (Th1) and Th2 cytokine expressing T-cell subpopulations in women with three or more recurrent spontaneous abortions (RSA), multiple implantation failures after in-vitro fertilization and embryo transfer (IVF/ET) (three or more) or during normal pregnancy. METHOD OF STUDY: Absolute cell counts and percentages of CD3+, CD3+/CD4+, and CD3+/CD8+ T-cell populations expressing intracellular cytokines [interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4 and IL-10] was studied by four-color flow cytometry in 15 RSA and 13 implantation failure patients. Eighteen fertile non-pregnant and 47 normal pregnant women were also compared with regard to intracellular cytokine expression. RESULTS: Interleukin-10 producing CD3+/CD8+ T-cell counts were significantly lower in women with RSA (P < 0.05) and implantation failures (P < 0.05), and TNF-alpha producing CD3+/CD4+ T-cell counts were higher in women with RSA (P < 0.05) and implantation failures (P < 0.005) than those of non-pregnant fertile controls. During normal pregnancies, first trimester IL-4 expressing CD3+, CD3+/CD4+ T-cell counts (P < 0.05) and IFN-gamma expressing CD3+ T-cell counts (P < 0.05) were significantly higher than those of third trimester (P < 0.05). First trimester TNF-alpha expressing CD3+/CD8+ T-cell counts were significantly higher than those of second and third trimester women (P < 0.05). However, there are no differences in cytokine expression between non-pregnant and first trimester pregnant women. CONCLUSION: Absolute counts of IFN-gamma, IL-4, and TNF-alpha expressing T cells decrease with the progress of gestation (third trimester) during normal pregnancies. In women with implantation failures, absolute cell counts of TNF-alpha expressing CD3+/ 4- cells reflects the presence of dominant Th1 immune response. A significantly increased Th1 cytokine expression may be the underlying immune etiology for reproductive failures.     

A dominant role for chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) in mediating chemotaxis of CRTH2+ CD4+ Th2 lymphocytes in response to mast cell supernatants

Human cultured mast cells, immunologically activated with immunoglobuin E (IgE)/anti-IgE, released a factor(s) that promoted chemotaxis of human CRTH2+ CD4+ T helper type 2 (Th2) lymphocytes. Mast cell supernatants collected at 20 min, 1 hr, 2 hr and 4 hr after activation caused a concentration-dependent increase in the migration of Th2 cells. The effect of submaximal dilutions of mast-cell-conditioned media was inhibited in a dose-dependent manner by ramatroban (IC50 = 96 nm), a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), but not by the selective TP antagonist SQ29548, implicating CRTH2 in mediating the chemotactic response of these Th2 cells. The effect of mast-cell-conditioned media was mimicked by prostaglandin D2 (PGD2) and this eicosanoid was detected in the conditioned media from activated mast cells in concentrations sufficient to account for the activity of the mast cell supernatants. Treatment of the mast cells with the cyclo-oxygenase inhibitor diclofenac (10 microm) inhibited both the production of PGD2 and the CRTH2+ CD4+ Th2-stimulatory activity, while addition of exogenous PGD2 to conditioned media from diclofenac-treated mast cells restored the ability of the supernatants to promote chemotaxis of these Th2 cells. The degree of inhibition caused by diclofenac treatment of the mast cells was concordant with the degree of inhibition of chemotactic responses afforded by CRTH2 blockade. These data suggest that PGD2, or closely related metabolites of arachidonic acid, produced from mast cells may play a central role in the activation of CRTH2+ CD4+ Th2 lymphocytes through a CRTH2-dependent mechanism.     

南宁地区41例平衡易位携带者的细胞遗传学分析

目的通过对2856例反复流产、不良孕产史、不孕不育等患者进行染色体结果调查,探讨染色体平衡易位携带者的临床效应。方法采用外周血淋巴细胞染色体培养技术,G显带,核型分析。结果41例染色体平衡易位携带者中,男性21例,女性20例,常一常染色体易位39例,其中包括3例罗伯逊易位;性一常染色体易位2例。主要临床表现为女性反复自然流产、不良孕产史等;男性少弱精子症及不育等。结论染色体平衡易位是导致反复流产、不良孕产史、男性少弱精子症、不育等的重要因素之一。对于这些有反复流产史、不良妊娠史、不孕不育的临床患者,进行外周血染色体核型分析十分必要。     

早期不明原因反复自然流产发生与蜕膜中免疫细胞的研究进展

人类妊娠被认为是一种半同种异体抗原移植，母胎间存在着某种免疫耐受机制来维持妊娠的进行，但目前为止这种免疫耐受机制尚不明确。大量的研究发现不明原因反复自然流产患者蜕膜中调节性T细胞的数量和功能均显著降低，表明调节性T细胞在避免胎儿免疫排斥中发挥着重要的作用。同时NK细胞作为早期妊娠蜕膜中的优势淋巴细胞亦对妊娠的维持起着重要的作用，不明原因反复自然流产患者蜕膜NK细胞数量和活性比正常妊娠妇女明显升高，同时CD56^＋CD16^＋／CD56^＋CD16^-NK细胞比例失衡。由此可见，妊娠早期不明原因反复自然流产的发生与蜕膜中淋巴细胞的异常表达相关，通过对这种复杂机制的研究可以为不明原因反复自然流产的预防和治疗提供依据。     

Pre-implantation endometrial leukocytes in women with recurrent miscarriage.

Immunohistochemistry was used to investigate the leukocyte populations in the endometrium of women suffering recurrent miscarriage. Mid-luteal phase endometrial biopsies were taken from 22 patients with idiopathic recurrent miscarriage and from nine women with normal obstetric histories. The samples were dated histologically and stained with a panel of monoclonal antibodies to identify leukocytes. The outcome of any pregnancy in subsequent cycles following the biopsy was determined. Similar numbers of cluster designation (CD)3(+) and CD8(+) cells were seen in both groups. However, CD4(+), CD14(+), CD16(+), CD56(+) and MHC class II(+) cells were significantly higher in the recurrent miscarriage group than in the controls. Two patients had B cells (CD22(+)) in their endometrium. No CD57(+) cells were seen in the controls; however, eight of the patients had a few CD57(+) cells present. Only two patients, both from the recurrent miscarriage group, had CD69(+) leukocytes in their endometrium. Patients who had miscarriages following endometrial biopsy had significantly more CD4(+), CD8(+), CD14(+), CD16(+), and CD56(+) leukocytes in their endometrium than either those who had live births or women with proven fertility. A different population of leukocytes was found in the pre-implantation endometrium from recurrent miscarriage patients as compared to those from fertile controls. These differences were accentuated in women who had a miscarriage subsequent to the biopsy compared with those who subsequently had a live birth.     

Prostaglandin D2 and reproduction

This review highlights recent studies investigating the role of prostaglandin (PG)D2 in reproduction. PGD2 induces sleep, allergic responses, inhibition of platelet aggregation, and relaxation of vascular and non-vascular smooth muscle, and has some roles in reproduction. Two types of PGD2 synthase are known. Lipocalin-type PGD synthase is present in cerebrospinal fluid, seminal plasma and may play an important role in male reproduction. Another PGD synthase, hematopoietic PGD synthase is present in the spleen, fallopian tube, endometrial gland cells, extravillous trophoblasts and villous trophoblasts, and perhaps plays an important role in female reproduction. Recent studies demonstrate that PGD2 is probably involved in multiple aspects of inflammation through its dual receptor systems, DP and CRTH2. CRTH2 but not DP is a chemo-attractant receptor for PGD2. Interestingly, CRTH2 is a most reliable marker for the detection of human T helper type 2 (Th2) and T cytotoxic type 2 (Tc2) cells, and the percentages of CRTH expressing CD4+-T cells and CD8+-T cells were significantly higher in the decidua especially at the implantation site, suggesting that Th2 and Tc2 cells recruit into the materno-fetal interface, in a PGD2-mediated manner. PGD2 has a very unique effect to inhibit antigen presentation by inhibition of dendritic cell (DC) migration through DP but not CRTH2. PGD2 might appear to contribute to the maintenance of pregnancy by controlling the Th1/Th2 balance and antigen presentation by DCs through its dual receptor systems, CRTH2 and DP.     

T helper 1 and 2 cytokine expression in women with recurrent spontaneous abortions and infertility of repeated implantation failures

Normal pregnancy is proposed to be a T helper (Th) 2 immune response. Th2 cells induce antibody mediated immunity and secrete cytokines such as IL-4, 5 and 10, which favors a successful pregnancy. A few studies are reported to investigate Th1 or Th2 immune responses in women with reproductive failures. Recent advances in flow cytometric analysis enable us to detect presence of Th1 or Th2 immune response by isolating T cell subpopulations and defining their characteristics of intracellular cytokine expression. Women with recurrent spontaneous abortions (RSA) and infertility of multiple implantation failures after IVF/ET have significantly higher Th1/Th2 ratios as compared to normal fertile women. Intracellular TNF-α expression, TNF-α/IL-4 and TNF-α/IL-10 ratios in CD3+/CD4+ T cells are significantly different in women with implantation failure who got pregnant, who failed to get pregnant and normal fertile women. When the absolute cell counts of cytokine expressing cells are examined, women with RSA and implantation failures demonstrated significantly higher TNF-α expressing CD3+/CD4+ T cells and lower IL-10 expressing CD3+/CD8+ T cells than those of normal fertile controls. In conclusion, women with RSA and infertility of implantation failures demonstrate a Th1 biased immune response. Further molecular genetic study of Th1 and Th2 cytokines may shed the light to understand possible etiologies for dysregulation Th1/Th2 immune responses.