过继转输胚胎抗原耐受T细胞诱导自然流产孕鼠母胎免疫耐受

探讨过继转输胚胎抗原耐受T细胞对小鼠自然流产模型妊娠预后及宿主孕鼠免疫细胞对父系抗原免疫耐受状态的影响。以CBA/J×BALB/c为正常妊娠模型 ,CBA/J×DBA/ 2为自然流产模型 ,将自然流产模型CBA/J孕鼠于孕 4d (着床期 )分别腹腔注射大鼠抗小鼠CD80和CD86mAb或大鼠同型IgG。于孕 9d ,应用免疫磁珠阴性分选三组孕鼠的脾脏T细胞 ,并将三组T细胞分别转输至孕 4d的CBA/J×DBA/ 2孕鼠。于宿主孕鼠孕第 9天 ,采用单向混合淋巴细胞反应分析宿主孕鼠脾脏免疫细胞对父系抗原的增殖能力 ,并用流式细胞术分析经父系抗原刺激的宿主孕鼠脾脏T细胞内IL 2表达水平。于孕 1 4d分别观察宿主孕鼠的胚胎吸收率。结果显示 ,过继转输胚胎抗原耐受T细胞和转输正常妊娠模型孕鼠的T细胞均可诱导宿主孕鼠脾脏免疫细胞对父系抗原的增殖能力及IL 2的表达显著下降 (P <0 0 5 ) ,孕 1 4d胚胎吸收率也显著下降 (P <0 0 5 )。这些结果表明 ,于孕早期过继转输胚胎抗原耐受T细胞和转输正常妊娠模型孕鼠的T细胞能诱导宿主孕鼠母 胎免疫耐受 ,防止母体对胚胎的免疫排斥 ,从而使自然流产模型的妊娠预后达到正常妊娠水平。     

过继转输的胚胎抗原耐受T细胞在妊娠小鼠体内细胞因子及协同刺激分子的表达特征

目的 :分析过继转输的小鼠胚胎抗原耐受T细胞内细胞因子及细胞表面协同刺激分子的表达特征。方法 :以♀CBA/J×♂DBA/ 2为自然流产模型 ,将自然流产模型CBA/J孕鼠于孕 4天 (着床期 )分别腹腔注射大鼠抗小鼠CD80和CD86mAb或大鼠同型IgG。于孕 9天 ,应用免疫磁珠阴性分选两组孕鼠的脾脏T细胞 ,将T细胞进行碳氧氢化荧光素双乙酸盐琥珀酰亚胺酯 (CFSE)体外荧光标记 ,再分别过继转输至孕 4天的CBA/J×DBA/ 2孕鼠。在宿主孕鼠孕第 9天 ,处死小鼠取脾细胞 ,用流式细胞术分析在DBA/ 2父系抗原刺激下过继转输的T细胞细胞因子IL 2、IL 4、IL 10和IFN γ及协同刺激分子CD2 8和CTLA 4的表达。结果 :与过继转输的胚胎抗原非耐受T细胞相比 ,过继转输的胚胎抗原耐受T细胞IL 10的表达显著增加 ,IL 2和IFN γ的表达则显著下降 (P <0 0 5 ) ,IL 4的表达无明显改变 (P >0 0 5 ) ;细胞表面CD2 8的表达显著下降 ,而CTLA 4的表达却显著增加 (P <0 0 5 )。结论 :过继转输的小鼠胚胎抗原耐受T细胞内Th2型细胞因子和表面CTLA 4表达上调 ,而Th1型细胞因子和表面CD2 8的表达则下降。胚胎抗原耐受T细胞通过Th2型细胞因子表达优势和协同刺激信号降调节 ,在母 胎免疫耐受的维持中发挥着重要作用。     

过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分析

目的 :观察过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分布 ,以探讨免疫耐受T细胞在诱导受体母 胎免疫耐受中的作用机制。方法 :于孕 4天 (着床期 )给小鼠自然流产模型CBA J×DBA 2孕鼠腹腔注射抗CD80和CD86mAb ,以诱导母 胎免疫耐受。孕 9天应用免疫磁珠分选孕鼠脾脏T、B细胞 ,并用CFSE体外荧光标记。将标记的T、B细胞分别转输至孕 4天的CBA J×DBA 2孕鼠 ,36小时后在双光子共聚焦显微镜下观察T、B细胞在受体体内脾脏、子宫引流淋巴结及母 胎界面组织中的分布。结果 :过继转输的T、B细胞分布在孕鼠体内脾脏和子宫引流淋巴结 ,但并不留驻在母 胎界面。结论 :过继转输的胚胎抗原耐受T细胞定居于外周免疫器官 ,从而介导受体孕鼠对相应父系抗原的免疫耐受。     

复发性自然流产育龄期妇女母-胎界面中细胞因子表达分析

目的:分析复发性自然流产(RSA)育龄期妇女母-胎界面中相关细胞因子表达水平变化。方法:连续选择近期就诊的RSA育龄期妇女21例,收集流产蜕膜组织,采用酶联免疫吸附法进行蜕膜组织液中IL-2、IL-4、IL-10、IFN-γ浓度等指标检测,并与同期早孕并行人工流产终止妊娠妇女(对照组,20例)相同测试结果比较。结果:RSA组蜕膜组织液IL-2和IFN-γ浓度均明显高于对照组,而IL-4和IL-10浓度则显著低于后者(P均<0.01)。结论:RSA育龄期妇女母-胎界面上存在着明确的Th1优势状态表达。     

过继转输正常妊娠孕鼠的T细胞抑制自然流产孕鼠对胚胎的排斥

目的　观察过继转输正常妊娠孕鼠的T、B细胞对小鼠自然流产孕鼠妊娠预后的影响。方法　以♀CBA J×♂BALB c为正常妊娠模型 ,♀CBA J×♂DBA 2为自然流产模型 ,于孕 9d ,采用单向混合淋巴细胞反应分析两组孕鼠脾脏免疫细胞对父系抗原的增殖能力及上清液IL 2分泌水平 ,以研究孕鼠母 胎免疫耐受状态 ;并于孕 14d观察两种模型的胚胎吸收率。然后 ,应用免疫磁珠阴性分选两组孕鼠脾脏T细胞和B细胞 ,并将两种细胞分别转输至孕 4d(着床期 )的♀CBA J×♂DBA 2孕鼠 ,于孕 14d分别观察宿主孕鼠的胚胎吸收率。结果　与自然流产孕鼠比较 ,正常妊娠孕鼠孕 9d脾脏免疫细胞对父系抗原的增殖能力及IL 2的分泌显著下降 (P <0 .0 5 ) ,孕 14d胚胎吸收率也显著下降 (P <0 .0 5 )。与转输自然流产孕鼠的T细胞相比 ,转输正常妊娠孕鼠T细胞的宿主孕鼠的胚胎吸收率显著下降 (P <0 .0 5 ) ;而转输两组B细胞的宿主孕鼠的胚胎吸收率差异无显著性 (P >0 .0 5 )。结论　于孕早期过继转输正常妊娠孕鼠的T细胞能诱导宿主孕鼠母 胎免疫耐受 ,防止母体对胚胎的免疫排斥 ,使自然流产模型的妊娠预后达到正常妊娠水平。     

米非司酮通过增强母-胎界面Th1型偏移导致流产

目的：探讨米非司酮对母胎界面Th1/Th2型细胞因子动态平衡的影响.方法：将63例早孕期妇女随机分为2组,一组一次服用米非司酮200 mg,另一组为对照组,收集其蜕膜组织.应用免疫组化法,评价Th1型细胞因子（IL-2、IFN-γ）、Th2型细胞因子（TGF-β2、IL-4）的表达.结果：正常妊娠时,在母-胎界面Th2型细胞因子（IL-4）以及TGF-β2的表达较高;Th1型细胞因子（IL-2、IFN-γ）的表达较低,尤其是IL-2.服用米非司酮后,蜕膜Th1型细胞因子（IL-2、IFN-γ）表达显著升高;而母-胎界面Th2型细胞因子（IL-4）以及TGF-β2的表达无明显变化.结论：米非司酮打破了正常妊娠时母胎界面Th2型免疫优势;显著升高Th1型细胞因子（IL-2、IFN-γ）表达,形成了Th1型免疫偏离,导致流产的发生.     

CD86协同刺激信号在孕早期母—胎界面Th1／Th2型细胞因子表达调控中的作用

目的探讨CD86协同刺激信号在孕早期母-胎界面Th1/Th2型细胞因子表达调控中的作用。方法建立正常妊 娠模型CBA×Balb/c和自然流产模型CBA× DBA/2。于孕第4、6、8、10天给CBA孕鼠腹腔注射大鼠IgG作为对照组;仅于孕第4 天或于孕第4、6、8、10天给CBA孕鼠腹腔注射大鼠抗小鼠CD86 mAb。孕第14天计算两种模型各实验组胚胎吸收率R。ELISA 法测定孕第9和第14天各实验组母-胎界面组织体外培养上清中Th1/Th2型及相关细胞因子(IL-2、IFN-γ、TNF-α、IL-4、IL-10、 TGF-β2)表达水平。结果孕早期干预CD86协同刺激信号,对正常妊娠模型母-胎界面孕第9和第14天IL-4、IL-10、TGF-β2以及 IFN-γ、TNF-α表达均无显著影响,其胚胎吸收率亦无显著变化。自然流产模型中,孕早期干预CD86协同刺激信号后,孕第9、 第14天母-胎界面IL-4、IL-10、TGF-β2表达均显著增加(P＜0.05);而IFN-γ、TNF-α表达显著下降(P＜0.05)。胚胎吸收率亦 显著下降(P＜0.05)。结论孕早期母-胎界面CD86协同刺激信号的调节紊乱可能是触发母体对胚胎产生免疫排斥的重要病 理因素,于孕早期干预CD86协同刺激信号能够恢复母-胎界面Th1型/Th2型免疫调节的生理平衡,从而诱导母-胎免疫耐受。     

NKT细胞在母胎界面免疫调节中的作用

NKT细胞(natural killer T cell)是一类新型的免疫调节细胞,该类细胞同时表达T细胞表面受体(TCR)和NK细胞的抗原NK1.1。NKT细胞上的TCR通过限制性识别由CD1d分子提呈的脂类抗原,进一步被激活后可分泌多种细胞因子,从而调节机体的免疫反应。近年来逐渐认识到NKT细胞可能参与了母胎界面的免疫调节。     

NK细胞在早孕期母-胎界面的功能

早孕期NK细胞在子宫蜕膜局部大量累积,蜕膜NK(decidual NK,dNK)细胞具有独特表型与功能。有研究显示,dNK细胞在子宫螺旋动脉重铸、局部免疫微环境维持和孕期母体抗病原体感染方面均发挥了重要作用,但其具体机制尚未阐明。文章综述近年来dNK细胞在胎盘血管重铸和母-胎免疫耐受方面的新发现。     

蜕膜巨噬细胞在母-胎界面的作用研究

妊娠早期,大量的免疫细胞在子宫局部聚集,母体对带有父系抗原的胎儿并不排斥,而是通过精细的母-胎免疫对话形成独特的母-胎免疫微环境。蜕膜巨噬细胞(decidual macrophage, DM)作为母-胎界面第二大类白细胞,在妊娠早期发挥抗炎和吞噬作用,并通过调节蜕膜局部免疫微环境、重塑蜕膜血管,参与胚胎植入和维持妊娠等过程。若DM表型和功能发生异常,将会导致早期妊娠失败或妊娠中、晚期并发症,其确切机制尚未阐明。文章着重论述妊娠早期DM的特点及其在正常妊娠建立和维持以及病理妊娠中的作用。     

Adoptive transfer of paternal antigen-hyporesponsive T cells facilitates a Th2 bias in peripheral lymphocytes and at materno-fetal interface in murine abortion-prone matings

PROBLEM: To investigate the Th1/Th2 cytokine changes in abortion-prone recipient mice adoptively transferred by the paternal antigen-hyporesponsive T cells. METHOD OF STUDY: The paternal antigen-hyporesponsive T cells were generated by the anti-B7 monoclonal antibody (mAb) treatment and adoptively transferred into pregnant CBA/J mice of abortion-prone matings on day 4 of gestation. The intracellular expressions of Th1 cell-derived cytokine, tumor necrosis factor-alpha, gamma-interferon and interleukin-2 (IL-2) and Th2 cell-derived cytokine, IL-4 and IL-10 in the maternal spleen were analyzed by flow cytometry, and secretions of the Th1 and Th2 cytokines in supernatant of the feto-placental unit culture were analyzed by an enzyme-linked immunosorbent assay. RESULTS: Our findings showed the increased secretion of Th1 cytokines and the decreased secretion of Th2 cytokines in abortion-prone matings. Treatment with anti-B7 mAbs on day 4 of gestation enhanced Th2 and reduced Th1 cytokine production in abortion-prone matings. Similarly, adoptive transfer of paternal antigen-hyporesponsive T cells induced maternal tolerance to the fetus and displayed a Th2 bias both in the peripheral lymphocytes and at the materno-fetal interface of the abortion-prone matings. CONCLUSIONS: These findings indicate that the Th2 cytokine bias and an increase in fetal viability induced by the anti-B7 mAb treatment can be transferred to other pregnant mice of the abortion-prone matings.     

TH1和TH2细胞在痢疾疫苗诱导小鼠产生粘膜免疫应答中的调节作用

目的　在初步了解ＣＤ４ ＋ Ｔ 细胞参与痢疾疫苗诱导小鼠肠粘膜免疫应答的基础上,从细胞及分子水平进一步探索ＴＨ１ 和ＴＨ２ 细胞在小鼠肠粘膜对痢疾疫苗免疫应答中的调节作用。方法　以本室构建的福氏２ａ 及宋内氏双价痢疾疫苗株ＦＳ２１１７ 或ＦＳ５４１６ 灌胃免疫小鼠,从细胞表型及几种细胞因子ｍ ＲＮＡ 表达水平两方面观察小鼠肠粘膜中Ｐａｙｅｒ 氏结（ Ｐｅｙｅｒ’ｓ ｐａｔｃｈ , ＰＰ） 及肠系膜淋巴结等免疫诱导部位ＴＨ１ 和ＴＨ２ 细胞的动态变化规律。结果　（１） 痢疾疫苗灌胃免疫后早期,小鼠肠粘膜的免疫诱导部位表现为ＴＨ１ 型细胞亚群百分率的显著降低;免疫后晚期ＴＨ１ 型细胞亚群百分率则显著升高,且高于未免疫的正常小鼠。（２） 痢疾疫苗免疫后从小鼠ＰＰ 中分离的ＣＤ４ ＋ Ｔ 细胞,在特异性痢疾疫苗抗原刺激下,先出现ＩＬ５ 、ＩＬ６ 的高表达,后出现ＩＦＮγ、ＩＬ２ 的高表达。结论　痢疾疫苗免疫后小鼠肠粘膜免疫的诱导部位早期表现为ＴＨ２ 型细胞亚群的优势活化,晚期逆转为ＴＨ１ 型细胞亚群的优势活化。     

The presence of blood in the transfer catheter negatively influences outcome at embryo transfer

BACKGROUND: Embryo transfer (ET) influences pregnancy rates in patients undergoing assisted reproduction. Data are conflicting as to which variables affect ET success. This study examines variables that may affect outcome after ET in assisted reproductive technology patients who had high-quality embryos transferred. METHODS: Over a 23 month period, 669 consecutive cycles were examined. Only patients having grade I and grade II embryos, or blastocyst transfers, were included in this retrospective analysis. A total of 584 consecutive cycles met study criteria. At the time of ET, the following variables were recorded: aborted first attempt at ET; presence of blood and/or mucus in or on the transfer catheter after ET; ease of ET as judged by provider; need for mock embryo transfer immediately before the actual transfer and retention of embryos in the transfer catheter. These variables were retrospectively analysed for their impact on implantation rate (IR) and clinical pregnancy rate (CPR). RESULTS: There were 290 gestations (49.7% CPR). Multiple attempts at ET, subjective difficulty of ET, performance of a sham pass immediately prior to embryo transfer, and presence of mucus on or in the catheter did not affect the CPR or IR. No difference was noted in the mean age of patients having or lacking any of these factors. There was a significant association between the presence of blood on or in the catheter and decreased IR (P = 0.015) and CPR (P = 0.004). Retained embryos also decreased IR (P = 0.03). Multivariable analysis confirmed that the presence of blood on the transfer catheter was the most important of these transfer characteristics in predicting IR (P = 0.042) and CPR (P = 0.018). CONCLUSIONS: These results suggest that when only high-grade embryos or blastocysts are transferred, the presence of blood on the catheter is associated with decreased IR and CPR in assisted reproduction.     

Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer

BACKGROUND: The study aim was to investigate the impact of the developmental stage of embryos on pregnancy outcome of frozen embryo transfer (FET). METHODS: The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between three cryopreservation strategies utilizing either zygote, day 2 or day 3 embryo freezing. RESULTS: A total of 4006 embryos was analysed in 1657 thaw cycles. The highest (P < 0.0001) survival rate (all cells survived) was observed for zygotes (86.5%), followed by day 2 (61.7%) and day 3 (43.1%) embryos. FET was performed in 1586 (95.7%) of all thaw cycles, resulting in overall clinical pregnancy and implantation rates of 20.7 and 14.2% respectively. The delivery rate per transfer was 16.5%, and live birth rate per transferred embryo 11%. There were no significant differences in clinical pregnancy, implantation, delivery and birth rates between frozen zygote, day 2 and 3 embryo transfers. However, an elevated miscarriage rate was observed in the day 3 group (45%) compared with zygotes (21.3%; P = 0.049) and day 2 embryos (18.3%; P = 0.004). The overall efficacy of FET (birth rate per thawed embryo) was 7.3%. The efficacy was lower in day 3 group (4.2%) than in the zygote (7.1%; P = 0.082) and day 2 (7.6%; P = 0.027) groups. CONCLUSIONS: The developmental stage of embryos at freezing has a profound effect on their post-thaw survival, but seems to have little effect on rates of clinical pregnancy, implantation, delivery and birth after FET. The elevated miscarriage rate for day 3 frozen embryo transfers may be caused by damage during freeze-thaw procedures. The low survival rate and elevated miscarriage rate were both responsible for a reduced overall efficacy for day 3 FET when compared with zygotes and day 2 embryos.     

The impact of the embryo transfer catheter on the pregnancy rate in IVF

BACKGROUND: The aim was to assess whether the type of embryo transfer set used for embryo transfer affects the ongoing pregnancy rate in IVF. METHODS: The TDT set was compared with the K-soft 5000 in a large, prospective, randomized study. Patients were randomized moments before transfer by drawing a consecutively numbered, sealed, opaque envelope indicating the catheter to be used. RESULTS: 2059 embryo transfers in 1296 patients were analysed. The ongoing pregnancy rate was significantly higher in the K-soft group. If the first transfer of a patient (n = 1296) within this study period was analysed, the ongoing pregnancy rates were 27.1 versus 20.5% (P = 0.006). If the analysis is limited to patients that underwent their very first transfer ever (n = 607), the ongoing pregnancy rates were 30.3 versus 20.0% (P = 0.003) in favour of the K-soft. CONCLUSION: We conclude from these data that the type of embryo transfer set used for embryo transfer does affect the ongoing pregnancy rate and that the impact of the variable transfer catheter on the ongoing pregnancy rate increases when the a priori chance of pregnancy increases.     

Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study

INTRODUCTION: In a randomized controlled trial, we assessed whether pregnancy outcome would be improved by extending embryo culture to day 5 and transferring a blastocyst in patients with at least four good-quality embryos on day 3. METHODS: Multifollicular ovarian stimulation was performed with a GnRH agonist in 44% of patients and with a GnRH antagonist in 56%. Overall, 164 patients younger than 37 years fulfilled embryo quality criteria (at least four having at least six cells on the morning of day 3, maximum 20% anucleate fragments) on the third day of culture and were randomized to the day 3 (n = 84) or day 5 (n = 80) groups. Equal numbers of embryos (n = 2) were transferred in each group. RESULTS: Demographics, stimulation parameters and embryological data were comparable in the two groups. Blastocyst-stage transfer resulted in a significantly higher ongoing pregnancy rate [51.3 versus 27.4%; odds ratio (OR) 2.78, 95% confidence interval (CI) 1.45-5.34] and live birth rate (47.5 versus 27.4%; OR 2.40, 95% CI 1.25-4.59) compared with day-3 embryo transfer. A high twin birth rate was observed in both groups (36.8 versus 30.4%; P > 0.05). CONCLUSIONS: A threshold of four good embryos on the third day of embryo culture appears to indicate that the patient will benefit from embryo transfer at the blastocyst stage and have a better chance of achieving a live delivery than with cleavage-stage embryo transfer.