人自体PBMC对反复种植失败患者子宫内膜容受性标志物及胚胎着床的影响

发布时间：2018-05-23 15:09

本文选题：外周血单个核细胞(PBMC) + 反复种植失败(RIF)　；参考：《郑州大学》2017年硕士论文

【摘要】：背景及目的由于不孕症发病率逐渐升高,辅助生殖技术(ART)迅速发展,体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)及其衍生技术使众多不孕症患者借助获得了后代[1]。其中一大部分夫妇在经历过几次IVF治疗后仍然不能成功妊娠,导致了严重的生活质量下降,并且每一次失败都会带来巨大的财务负担。反复着床失败(repeated implantation Failure,RIF)已成为辅助生殖领域研究的热点和难点[2]。胚胎成功着床的关键因素在于母体和胚胎之间的良好对话[3],因此如何改善胚胎质量、提高子宫内膜容受性,促进母胎对话和免疫耐受成为了辅助生殖领域治疗RIF的主要研究方向。最近Yoshioka等[4]首先报道了对反复着床失败患者进行宫腔灌注人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC),结果显示反复着床失败患者的临床妊娠率、着床率和活产率得到了显著提高。PBMC主要包括T淋巴细胞、B淋巴细胞和单核细胞。PBMC分泌的IL-1α、IL-1β和TNF-α等细胞因子在胚胎着床中发挥着重要作用[5]。其机制可能与PBMC分泌的多种炎性因子在内膜容受性上调和提高胚胎侵袭力有关。本课题通过体外培养实验和临床随机对照试验研究:1.体外培养条件下自体PBMC对RIF患者子宫内膜容受性标志物人白血病抑制因子LIF和整合素αVβ3表达的影响;2.PBMC对胚胎表面LIF受体表达的影响;3.研究自体PBMC宫腔灌注对RIF患者行冻融胚胎复苏移植(Frozen-thawed embryo transplantation,FET)助孕妊娠结局的影响。研究方法1、体外培养条件下自体PBMC对RIF患者子宫内膜容受性标志物LIF和整合素αVβ3表达的影响1)选择2015年5月至2016年5月于郑州大学第二附属医院反复种植失败患者12例,使用内膜取样器收集患者月经第14天子宫内膜组织,分离子宫内膜腺上皮和间质细胞,体外培养传至2-3代后冷冻保存。同时收集其外周血使用Ficoll分离液分离出PBMC细胞。2)以1×105个/ml密度将子宫内膜细胞接种于四孔板,37℃,5%CO2培养24h后,更换含雌孕激素培养液1ml,加雌孕激素培养液当日记作Day0。3)体外培养至Day2时,实验组加入以密度为3×106/ml的PBMC细胞悬液0.5ml。对照组加入等量培养液。继续体外培养至Day6。4)隔日换液,并分别于Day1、3、5收取培养上清液待测。5)应用酶联免疫法检测培养液中LIF和整合素αVβ3的浓度。2、PBMC对体外胚胎和子宫内膜共培养模型中胚胎表面LIF受体表达的影响1)收集签署废弃胚胎知情同意书的胚胎,其中Day3胚胎共58枚。2)用含雌孕激素的培养液体外培养子宫内膜细胞,模拟人体子宫内膜环境,加雌孕激素培养液当日记作Day0。3)A组(实验组):培养至Day2时,观察子宫内膜贴壁情况,实验组加入以密度为3×106/ml的PBMC细胞0.5ml,Day3加入胚胎,共24枚,每孔2枚,养至Day6,观察囊胚形成情况。4)B组(对照组):培养至Day2时添加等量培养液,Day3加入胚胎,共24枚,每孔2枚,养至Day6,观察囊胚形成情况。5)C组(普通胚胎组):共10枚,不进行培养,将胚胎复苏后直接进行检测。6)应用免疫荧光方法检测细胞期胚胎和囊胚表面LIFR的表达情况。3、自体PBMC宫腔灌注对RIF患者FET妊娠结局的影响1)研究对象:选取2015.1月至2016.12来我院生殖中心就诊的符合反复种植失败诊断的患者。2)分组情况:将符合纳入条件的患者根据就诊时间排序并编号后,应用随机数表法进行随机分组,随机分为实验组和对照组,截至2016年12月,共入组78例,实验组A(56例)和对照组B(22例)。设置双盲,研究对象和主治医师均不知情分组情况。3)实验组于主导卵泡破裂日或服用雌激素内膜厚度达到标准后进行孕酮转化当日抽取自体外周血4 ml,分离PBMC,方法同第一部分,分别于移植前1天、3天B超引导下用胚胎移植管将200ul PBMC细胞悬液轻轻注入宫颈内口上方。适时胚胎移植,保胎治疗。对照组宫腔灌注等量培养液,适时胚胎移植并保胎移植。4)胚胎移植35日后行彩超检查,超声下看到孕囊胎芽为临床妊娠。结果1、体外培养条件下自体PBMC对RIF患者子宫内膜容受性标志物LIF和整合素αVβ3表达的影响1)培养第3天两组培养液中LIF浓度无明显差异(P0.05),培养第5天时,与单独子宫内膜组相比,共培养组培养液中LIF浓度明显升高(P0.05)。2)培养第3天两组培养液中整合素αVβ3浓度无明显差异(P0.05),培养第5天时,与单独子宫内膜组相比,共培养组整合素培养液中αVβ3浓度明显升高(P0.05)。2、PBMC对体外胚胎和子宫内膜共培养模型中胚胎表面LIF受体表达的影响1)本研究共检测D3胚胎10枚,为4/II—9/II,对照组囊胚10枚,级别为3BB-5BB,实验组共获得12枚囊胚,级别为3BB-5BB。实验组和对照组囊胚形成率分别为:50.0%,41.7%,差异无统计学意义(P0.05)。2)三组免疫荧光OD值差异显著P0.05;两两比较结果显示:LIFR的免疫荧光OD值实验组囊胚对照组单独胚胎组,P均0.05。3、自体PBMC宫腔灌注对RIF患者行FET助孕的妊娠结局1)PBMC治疗组和对照组的平均年龄、不孕年限、不孕类型、既往移植周期数、既往移植优胚数等相比,均无统计学差异(P0.05)。2)PBMC治疗组的胚胎种植率和临床妊娠率均高于对照组(P0.05)。结论1.自体PBMC可以提高RIF患者子宫内膜细胞体外培养条件下LIF和整合素αVβ3的浓度,进而上调子宫内膜容受性。2.PBMC可以促进子宫内膜共培养条件下的胚胎表面LIF受体的表达。3.自体PBMC宫腔灌注可以提高RIF患者行FET助孕的胚胎种植率和临床妊娠率。
[Abstract]:Background and objective the incidence of infertility is increasing, and assisted reproductive technology (ART) develops rapidly. In vitro fertilization and embryo transfer (in vitro fertilization and embryo transfer, IVF-ET) and its derivatization technology, many infertile patients with the aid of acquired offspring [1]. are still unable to be treated several times after IVF. Successful pregnancy leads to a serious decline in the quality of life and a huge financial burden for every failure. Repeated implantation Failure (RIF) has become a hot and difficult point in the research of assisted reproductive fields. The key factor in the successful implantation of [2]. embryos is the good dialogue between the mother and the embryo [3], so the key factor is the [3] between the mother and the embryo. How to improve the quality of the embryo, improve the receptivity of the endometrium, promote the maternal fetal dialogue and immune tolerance have become the main research direction in the treatment of RIF in the assisted reproductive field. Recently, Yoshioka and other [4] first reported the intrauterine perfusion of human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) for patients with repeated implantation failure. The clinical pregnancy rate, implantation rate and survival rate of patients with repeated implantation failure have been significantly improved..PBMC mainly includes T lymphocytes, IL-1 alpha, IL-1 beta and TNF- a secreted by B lymphocytes and mononuclear cells, IL-1 beta and TNF- alpha, which play an important role in embryo implantation and the mechanism may be associated with a variety of inflammatory factors secreted by PBMC. In vitro culture experiment and clinical randomized controlled trial study: 1. the effect of autologous PBMC on the expression of human leukemia inhibitory factor LIF and integrin alpha V beta 3 in endometrium receptive markers in patients with RIF in vitro, and the effect of 2.PBMC on the expression of LIF receptor on the embryo surface; 3 The effect of autologous PBMC intrauterine perfusion on the outcome of pregnancy induced pregnancy in RIF patients (Frozen-thawed embryo transplantation, FET). 1, the effect of autologous PBMC on the endometrial receptive markers LIF and the expression of integrin alpha V beta 3 in RIF patients under in vitro culture, 1) selected from May 2015 to May 2016. 12 cases of recurrent failure in the Second Affiliated Hospital of Zhengzhou University were used to collect endometrium and endometrium for fourteenth days in patients with endometrium, and the endometrial gland epithelium and interstitial cells were isolated and cultured in vitro to the 2-3 generation for cryopreservation. At the same time, the peripheral blood was collected and separated out of PBMC cell.2 by Ficoll separation solution. 1 x 105 /ml densities were collected. After inoculating endometrium cells into four orifice plates, 37 degrees C, 5%CO2 culture for 24h, and replacement of estrogen and progesterone culture solution 1ml, and estrogen and progesterone culture as Day0.3) in vitro culture to Day2, the experimental group added a PBMC cell suspension 0.5ml. control group with a density of 3 x 106/ml, and continued in vitro culture to Day6.4). The concentration of LIF and integrin alpha V beta 3 in the culture medium was detected by enzyme linked immunosorbent assay (.5), respectively,.2, PBMC on the expression of LIF receptor on the embryo surface in the co culture model of the embryo and endometrium 1) to collect the embryos of the informed consent form of the abandoned embryo, of which 58.2 in Day3 embryos were used to contain females. The endometrial cells were cultured outside the liquid of progesterone, the endometrial environment was simulated in the human body, and the estrogen and progesterone culture fluid was used as a diary of Day0.3) A group (experimental group). When cultured to Day2, the wall of the endometrium was observed. The experimental group was added to the PBMC cell 0.5ml of 3 x 106/ml, and Day3 was added to the embryo, 24, 2 of each hole, and observed to Day6. The formation of blastocysts (.4) group B (control group): adding equal amount of culture to Day2, Day3 adding embryos, 24 pieces, 2 holes, Day6,.5 of the blastocyst formation, C group (common embryo group): 10, no culture, and immediately after the embryo resuscitation to detect.6) using immunofluorescence method to detect the LIF cell embryos and blastocyst surface LIF The expression of R.3, the effect of autologous PBMC intrauterine perfusion on the outcome of FET pregnancy in RIF patients 1) the study was to select the cases of.2 in the patients who were diagnosed with repeated implantation failure in the reproductive center of our hospital from 2015.1 months to 2016.12: the random number table was used after the eligible patients were sorted and numbered according to the time of treatment. Groups were divided into experimental group and control group randomly. As of December 2016, 78 cases were enrolled in the group, A (56 cases) and control group B (22 cases) in the experimental group. The experimental group was set double blind, the research subjects and the chief physicians were unaware group.3). The experimental group was taken on the day of the leading follicle rupture day or the estrogen intima thickness reached the standard and took the autologous on the day of progesterone transformation. The peripheral blood was 4 ml, and the PBMC was separated from the first part. 1 days before the transplantation, the 200ul PBMC cell suspension was gently injected into the upper cervix of the cervix under the guidance of the embryo transfer tube on 3 days. The results were 1, the effect of autologous PBMC on the endometrial receptive marker LIF and the expression of integrin alpha V beta 3 in RIF patients was 1). There was no significant difference in LIF concentration in two groups of culture third days (P0.05). The culture medium of co culture group was compared with the individual endometrium group. There was no significant difference in the concentration of integrin alpha V beta 3 in the medium of medium LIF (P0.05).2). The concentration of alpha V beta 3 in the culture fluid of the co culture group was significantly increased (P0.05).2 and PBMC to the LIF receptor on the surface of the embryo and the endometrium culture in the culture medium of the co culture group at fifth days. The concentration of integrin alpha V beta 3 was not significantly different (P0.05) in the culture third days. 1) 10 D3 embryos were detected in this study, 4/II 9/II, 10 blastocysts in the control group and 3BB-5BB, and 12 blastocysts were obtained in the experimental group. The formation rate of the blastocyst in the experimental group and the control group was 50%, 41.7%, the difference was not statistically significant (P0.05).2) and the difference between the three groups of immunofluorescence was significant P0.05; 22 comparative knot. The results showed that the LIFR immunofluorescence o d experimental group had a single embryo group of blastocyst control group, P was 0.05.3, and the autologous PBMC uterine cavity perfusion was 1 for the FET pregnancy outcome of RIF patients. The average age of the PBMC treatment group and the control group, the number of infertility, the type of infertility, the number of previous transplantation cycles, and the number of previously transplanted embryos, were not statistically different (P0.05).2). The embryo implantation rate and clinical pregnancy rate in the PBMC treatment group were higher than those of the control group (P0.05). Conclusion 1. autologous PBMC can improve the concentration of LIF and integrin alpha V beta 3 under the culture conditions of endometrium in RIF patients, and then up regulation of endometrial receptive.2.PBMC can promote the expression of LIF receptor on the surface of endometrium under the condition of co culture. .3. autologous PBMC intrauterine perfusion can improve the embryo implantation rate and clinical pregnancy rate of FET in RIF patients.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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