Decidual natural killer cells in recurrent spontaneous abortion with normal chromosomal content.

PROBLEM: The maternal local immune responses in unexplained recurrent spontaneous abortion (RSA) are not yet well known. Maternal peripheral and decidual natural killer (NK) cells were evaluated in RSA with normal chromosomal content. METHOD OF STUDY: Maternal peripheral blood, villous trophoblast, and decidua were taken from 15 normal pregnancies and 9 RSA patients with normal chromosomes. The NK cells in decidual lymphocytes were evaluated by flow cytometry using monoclonal antibodies for CD56, CD16, and CD3. RESULTS: The percentages of CD56+ CD16- CD3- cells in decidual lymphocytes in RSA were lower than in normal pregnancies (P < 0.002). The CD56+CD16+/CD56+CD16- cells ratio in RSA was higher than in normal pregnancies (P < 0.02). CONCLUSION: The lower percentages of CD56+CD16-CD3- cells in RSA cases may show an inappropriate accumulation of NK cells in the decidua, and this finding may be a factor involved in RSA.     

Accumulation of CRTH2-positive T-helper 2 and T-cytotoxic 2 cells at implantation sites of human decidua in a prostaglandin D(2)-mediated manner

T-helper (Th) 2-type cytokines predominate in decidua, plausibly accounting for protection of a semiallograft, the embryo and placenta, from attack by the maternal immune system. However, localization of Th2 and T-cytotoxic (Tc) 2 cells in decidua has not been reported, presumably because of the difficulty in detecting intracellular cytokines in tissues. Here, by staining tissues for a novel surface maker of Th2/Tc2, the chemoattractant receptor-homologous molecule CRTH2, which is expressed on Th2 cells, we show that CRTH2(+) Th2 cells and CRTH2(+) Tc2 cells are significantly increased at the materno-fetal interface (implantation site) in decidua. We also show that trophoblast, uterine epithelium and endometrial glands all express haematopoietic-type prostaglandin (PG) D(2) synthase (hPGDS). Since CRTH2 is a chemoattractant receptor for PGD(2) and mediates PGD(2)-dependent migration of blood Th2 cells, our findings suggest that Th2 and Tc2 cells may be recruited to the materno-fetal interface, at least in part in a PGD(2)-mediated manner.     

Decrease in a specific killer cell immunoglobulin-like receptor on peripheral natural killer cells in women with recurrent spontaneous abortion of unexplained etiology

PROBLEM: The aim of this study was to investigate immunophenotypic characteristics of natural killer (NK) cells by assessing specific molecules expressed in women with recurrent spontaneous abortion (RSA) of unexplained etiology. METHOD OF STUDY: Peripheral blood cells were obtained from 20 RSA women and 15 fertile controls. The expression of perforin, CD94, CD161, CD158a, CD158b, and CD244 on CD3- CD56+ NK cells was analyzed by flow cytometry. RESULTS: A significant decrease in CD158a expression was demonstrated in RSA women (mean +/- SD, 22.9 +/- 8.7%) as compared with that in controls (33.6 +/- 15.7%) (P < 0.05). The percentage of NK cells showing dual expression of CD94 and CD161 was relatively higher in RSA women (55.1 +/- 10.2%) than in the controls (47.1 +/- 19.0%), but without statistically significant (P = 0.096). The expression of perforin, CD158b, or CD244 in RSA women did not differ from that in the controls. CONCLUSIONS: A divergence of the specific NK cell repertoire might be related to the etiology of RSA.     

Different expression of NOD2 in decidual stromal cells between normal and unexplained recurrent spontaneous abortion women during first trimester gestation

The NOD2 gene, encoding intracellular paternal recognition receptor (PRR) also called caspase activation and recruitment domain 15 (CARD15), is mutated in Crohn’s disease, an autoimmune-disorder. Unexplained recurrent spontaneous abortion (URSA) involved in complex auto-immune disorder. However, little is known about the expression of NOD2 protein at maternal-fetal interface with URSA patients. Our aim was to compare the expression levels of NOD2 in the decidual stromal cells (DSCs) from patients with normal pregnancy to those with unexplained recurrent spontaneous abortion (URSA) during first trimester pregnancy. Tissues and DSCs were collected from 12 patients with URSA and 26 patients with normal pregnancies that required abortion. DSCs in the normal pregnancy group showed significantly higher mRNA and protein levels of NOD2 than those isolated from the URSA group using real time PCR and in cell western. The appropriate expression of NOD2 in normal DSCs suggests that this protein may be required to sustain normal pregnancy.     

原因不明复发性流产患者调节性T细胞数量功能的改变及机制探讨

目的探讨原因不明复发性流产(URSA)发生的免疫机制,了解调节性T细胞在其发病中的作用。方法流式细胞术检测23例原因不明复发性流产(A组)、20例正常妊娠妇女(B组)和15例健康成年女性(C组)中CD4+CD25+T淋巴细胞的比例。免疫磁珠法分离CD4+CD25+细胞,逆转录聚合酶链反应(RT-PCR)法检测FOXP3的表达,酶联免疫吸附试验(ELISA)检测白细胞介素-10的水平。结果三组CD4+CD25+细胞比例无显著差异(P>0.05)。A组CD4+CD25+细胞表达FOXP3及分泌IL-10均低于B组和C组,具有显著性差异(P<0.05),而B组和C组表达FOXP3及分泌IL-10的水平无显著差异(P>0.05)。结论URSA患者调节性T细胞在外周血淋巴细胞中的比例虽无明显改变,但其功能明显减弱,可能是导致其发病的原因之一。     

Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion

It has been proposed that successful pregnancy is a T helper 2-type phenomenon, and that T helper (Th)1-type reactivity is deleterious to pregnancy. The objective of this study was to compare the concentrations of Th1 and Th2 cytokines produced by peripheral blood mononuclear cells from women undergoing unexplained recurrent spontaneous abortion (RSA) with those produced during normal pregnancy at a similar gestational stage. The control group consisted of 24 women with a history of successful pregnancies and the abortion group comprised of 23 women with a history of unexplained RSA. Blood from the control group was obtained at the end of the first trimester as gestational age controls for the abortion group from whom blood was collected at the time of abortion. Phytohaemagglutinin-stimulated peripheral blood cell culture supernatants were analysed for concentrations of cytokines. Significantly higher concentrations of Th2 cytokines were produced by the first trimester normal group than by the RSA group, while significantly higher concentrations of Th1 cytokines were produced by the abortion group as compared to first trimester normal pregnancy, indicating a distinct Th2-bias in normal pregnancy and a Th1-bias in unexplained RSA.     

The role of thrombophilia and thyroid autoimmunity in unexplained infertility, implantation failure and recurrent spontaneous abortion

BACKGROUND: The role of thrombophilia and thyroid autoimmunity in unexplained infertility (UI), implantation failure (IF) and recurrent spontaneous abortion (RSA) is controversial and poorly understood. METHODS: From March, 2004 to January, 2007, 119 women were prospectively included: 32 oocyte donors, 31 patients with UI, 26 with IF and 30 with RSA. The IF and RSA groups presented normal preimplantation genetic screening. Protein C, protein S, antithrombin III, lupus anticoagulant, activated protein C resistance (APCR), immunoglobulin M and G anticardiolipin antibodies, homocystine, Factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate reductase C677T mutation, thyroid-stimulating hormone (TSH), free thyroxine, anti-thyroid peroxidase (TPO) and anti-thyroglobulin (TG) antibodies were assessed. RESULTS: The prevalence of thrombophilia was high and similar among groups. In the IF group, the prevalence of APCR (15.4%), lupus anticoagulant (11.5%) and combined thrombophilia (19.2%) was higher, but not significantly different, than the other three groups. The prevalence of thyroid autoimmunity in women with IF (anti-TPO antibodies, P = 0.009; anti-TPO plus anti-TG antibodies,P = 0.04) and UI (anti-TPO, P = 0.002; anti-TG, P = 0.019; anti-TPO plus anti-TG antibodies, P = 0.005) was significantly increased in comparison to those with RSA. There was also a trend towards a higher prevalence of thyroid autoimmunity in the UI and IF groups than in the control group. TSH and free thyroxine levels all remained within a normal range. CONCLUSIONS: When embryo aneuploidy is ruled out, thrombophilia could constitute an etiologic factor in IF. Furthermore, thyroid autoimmunity is strongly related to UI and IF.     

Tim-3分子表达异常与原因不明复发性流产的关系

目的探究T细胞免疫球蛋白粘蛋白分子3（Tim-3）表达异常与不明原因复发性流产（URSA）的关系。方法选择31例URSA患者作为研究组,并以同期30例正常孕妇作为对照组。采用流式细胞术检测两组外周血单个核细胞（PBMC）中CD4＋Tim-3＋细胞/CD4＋细胞的比例,Western Blot技术检测蜕膜中Tim-3蛋白的相对含量,并以免疫磁珠法分选CD4＋Tim-3＋细胞,荧光定量RT-PCR检测两组外周血PBMC、CD4＋Tim-3＋细胞及蜕膜组织中Tim-3 mRNA的表达。结果 URSA组中,PBMC中CD4＋Tim-3＋细胞/CD4＋细胞为5.23±0.96%,蜕膜中Tim-3蛋白的相对表达量为0.64±0.09,PBMC及蜕膜中Tim-3 mRNA的相对表达量为0.65±0.09及0.50±0.07,均显著高于正常对照组（P〈0.01）。但是,URSA组CD4＋Tim-3＋细胞（Th1细胞）Tim-3 mRNA的表达量低于正常对照组,差异具有显著性（P〈0.01）。结论 Th1细胞Tim-3表达降低,Th1细胞过度活化可能参与了URSA的发生发展过程。     

MTHFR基因C677T位点多态性与不明原因重复性自然流产的相关性

目的　检测不明原因重复性流产 (unexplained recurrent spontaneous abortion,URSA)患者亚甲基四氢叶酸还原酶 (methylenetetrahydrofolate reductase,MTH FR)基因 C6 77T位点多态性 ,探讨其与血清同型半胱氨酸、叶酸、维生素 B1 2 及与不同临床特征的关系。　方法　 U RSA非孕期患者 5 7例 ,对照组 5 0名 ,空腹血清总高半胱氨酸 (homocysteine,Hcy)的检测采用高压液相色谱法 ,血清叶酸、维生素 B1 2 的检测采用放射免疫法。MTH FR基因 C6 77T多态性采用聚合酶链反应 -限制性片段长度多态性技术检测。结果　 URSA患者组 C/ C基因型频率显著低于正常对照组者 ,总的突变 T等位基因频率显著高于对照组。MTHFR T/ T基因型患者血清 Hcy水平显著增高 ,血清叶酸水平显著降低 ,MTHFR C6 77T基因型在不同年龄、地区、流产时间、流产性质患者的分布差异无显著性。流产 3次以上 (包括 3次 )的患者 6 77T/ T基因型、携带 T等位基因的 C/ T T/ T基因型频率均大于流产次数少于 3次的患者。　结论　 MTH FRC6 77T基因多态性是 URSA发病的遗传风险因素。     

T辅助细胞、NK细胞与原因不明复发性自然流产关系的研究进展

目前母-胎界面的免疫耐受机制是国内外的研究热点之一,URSA所表现的母-胎界面免疫耐受异常与T辅助细胞及NK细胞有密切的关系。本文首先对T辅助细胞和NK细胞的免疫状态、免疫耐受机制以及它们与URSA之间的关系进行了阐述,最后对目前与二者有关的URSA的主动免疫治疗研究进展作一综述。     

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。     

Galectin-1 reduction and changes in T regulatory cells may play crucial roles in patients with unexplained recurrent spontaneous abortion

To investigate the changes of Galectin-1 and T-lymphocyte phenotypes in unexplained recurrent spontaneous abortion (URSA). Totally 60 participants were recruited and divided into 3 groups in average: pregnant patients with URSA (URSA group), normal early pregnant women with induced abortion (IA group) and normal non-pregnant women (control group). After the tissue and blood sample were collected, Galectin-1 was measured using enzyme-linked immunosorbent assay. Then the proportion of T regulatory cells was determined by flow cytometry. The expression levels of Galectin-1 in IA group and URSA group was significantly higher than that in the control group (24.30 ± 3.06 and 6.23 ± 2.41 vs. 1.30 ± 0.66, P < 0.05). Besides, the expression level of Galectin-1 in URSA group was lower than that in IA group (P < 0.05). The percentage of CD4⁺CD25⁺Foxp3⁺ Tregs was lower in URSA group than IA group (0.77 ± 0.31 vs. 1.00 ± 0.35, P < 0.05) and the ratio of CD4⁺CD25⁺Foxp3⁺/CD4⁺ in URSA group was also obviously lower than that in IA and control group (P < 0.05). Galectin-1 and CD4⁺CD25⁺Foxp3⁺ may play essential roles in maintaining a normal pregnancy and their reduction may involve in the pathogenesis of URSA.     

TIM-3-Gal-9信号途径在原因不明复发性流产中的研究进展

体内免疫微环境中CD4+Th细胞亚群间平衡对维持正常妊娠的母胎耐受起关键作用,一旦这种格局被打破将导致原因不明复发性流产(URSA)的发生,然而具体机制未明。TIM-3是一个负调控分子,与其配体Gal-9结合诱导T细胞凋亡,抑制Th1和Th17免疫应答,对机体CD4+Th细胞亚群间平衡和免疫耐受调控起重要作用。TIM-3和Gal-9在URSA外周血和脱膜组织中有不同表达,TIM-3-Gal-9信号途径对正常妊娠的免疫平衡调节起重要作用,但机体机制知之甚少。本文就TIM-3-Gal-9信号途径对URSA免疫失衡和母胎耐受调控的研究进展作一综述,为URSA的防治提供新靶点和新思路。     

T and B lymphocyte subsets in patients with unexplained recurrent spontaneous abortion: IVIG versus placebo treatment

PROBLEM: To investigate circulating lymphocyte subsets in women with recurrent spontaneous abortion (RSA) in relation to pregnancy outcome and to treatment with intravenous immunoglobulin (IVIG). METHOD OF STUDY: Forty-one women with a history of unexplained RSA were examined during first trimester of pregnancy before IVIG or placebo treatment and after pregnancy. The results were compared with five healthy, non-pregnant women and five women in the first trimester of normal pregnancy. Circulating lymphocyte subsets with focus on T-cell subpopulations were determined by flow cytometry. RESULTS: The proportions of human leukocyte antigen (HLA)-DR positive T cells (CD3+ HLA-DR+), T-killer/effector cells (CD8+ S6F1+) and B cells (CD19+) were increased, whereas the proportion of T-suppressor/inducer cells (CD4+ CD45RA+) was decreased during first trimester pregnancy of RSA women compared with pregnant normal controls. T and B lymphocyte subsets did not correlate with pregnancy outcome on either IVIG or placebo group. CONCLUSIONS: In RSA patients, the immune system seems to be activated in contrast to the suppression noted in normal pregnancy.     

多囊卵巢综合征伴不明原因复发性流产患者外周血免疫调节基因表达谱的差异分析

目的从基因表达水平探讨多囊卵巢综合征(PCOS)发生不明原因反复自然流产的免疫调节机制。方法选择于我院就诊、按照鹿特丹标准诊断为PCOS,并发生2次或2次以上不明原因复发性流产的3例研究对象为病例组;同期就诊的月经规律、无异常妊娠史并已正常生育女性3例为对照组。提取2组样本的RNA进行免疫调节基因差异表达测序,并使用流式细胞术对2组的B细胞、T细胞及NK细胞及其亚型进行检测,对差异表达基因进行聚类分析及筛选统计,探讨可能参与致病机制的信号通路。结果共筛选出差异表达基因1 689个,通过GO富集分析,结合淋巴细胞亚群分析结果,筛选出14个免疫细胞功能相关的聚类;存在显著差异表达的基因主要富集在7个免疫调节功能相关的KEGG信号通路中。结论 PCOS患者存在免疫调节基因表达差异,可能通过调节NF-?B信号传导途径和B、T细胞受体信号通路导致复发性流产的发生。     

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.     

原因不明反复自然流产不同疗程的主动免疫治疗疗效观察

目的比较不明原因反复自然流产主动免疫治疗不同疗程的临床疗效。方法将160例APLA阴性患者分为3组,60例治疗组只治疗一疗程4次后妊娠,78例对照组每3周治疗1次,直至APLA转阳后妊娠;22例未治疗后直接妊娠,观察3组妊娠结局。APLA采用淋巴细胞毒实验进行检测。结果免疫治疗前APLA阳性率为8.57%,免疫治疗后治疗组、对照组APLA阳性率分别为41.67%、94.87%,治疗前后APLA比较差异显著（P〈0.01）。治疗组妊娠成功率为88.64%,对照组妊娠成功率为90.77%,两组比较差异不显著（P〉0.05）,未治疗组妊娠成功率为43.75%,与前两组比较差异显著（P〈0.01）。结论免疫治疗能有效刺激母体产生封闭抗体。对不明原因反复自然流产患者孕前进行一疗程4次治疗具有疗程短、疗效显著、妊娠结局好等特点。     

Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion.

The objective of this study was to establish whether or not patients with unexplained recurrent abortion have an increased incidence of haemostatic or metabolic abnormalities. Fifty-two patients with a history of unexplained habitual abortion (two or more spontaneous abortions before 16 weeks' gestation) were tested for protein S, protein C and antithrombin (AT) III deficiency, activated protein C (aPC) resistance, hyperhomocysteinaemia and anticardiolipin antibodies (ACA). The control group consisted of 67 healthy women with a history of only uncomplicated pregnancies. Blood samples were taken for measuring protein S, protein C, AT III, ACA and activated protein C resistance and a methionine loading test was performed. Of the 46 patients tested for protein S deficiency, 8 (17.4%) were positive. Of the 43 patients tested, two (4.7%) were protein C deficient and none was AT III deficient. Of the 42 patients tested for ACA, eight (19.1%) had detectable antibodies. Of the 44 patients tested for aPC resistance, two (4.6%) were positive. Finally, 35 patients were tested for hyperhomocysteinaemia and six (17.1%) were positive. It was concluded that parous women with a history of unexplained recurrent abortion have an increased incidence of hyperhomocysteinaemia and a trend of increased incidence of ACA can be found.