人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

子宫内膜异位症子宫内膜细胞对鼠胚体外早期发育的影响

子宫内膜异位症（EMs,内异症）与妇女不育有着密切的关系,约有25％～40％的不孕妇女患内异症,而内异症患者中,不育症约占30％～40％。为探讨子宫内膜异位症患者的子宫内膜是否对胚胎的发育产生不利的影响,本实验将两细胞期鼠胚与内异症妇女子宫内膜细胞体外共培养,观察其对鼠胚早期发育的影响。     

着床研究模型:离体胚泡与子宫内膜“共培养”

本工作用小鼠和豚鼠的离体胚泡与子宫内膜共培养方法,为研究着床与抗着床机理提供了一个体外研究模型。子宫内膜取自妊娠小鼠第5天上午8时前和妊娠豚鼠第4天下午,分别与小鼠或豚鼠胚泡共培养。经培养2～3天后可见胚泡从透明带逸出并附着于子宫内膜,部分植入内膜。     

子宫内膜细胞的凋亡

凋亡是一种选择性的细胞清除过程，有维持再生组织自身稳定性的作用。在月经周期的子宫内膜及胚泡着床后由其转化的蜕膜都有细胞凋亡，许多刺激因素可诱导细胞凋亡，基因的表达调节着细胞的敏感性。本文不子宫同膜细胞凋亡的分子生物学机制及其作用进行综述。     

子宫内膜异位症患者在位子宫内膜的研究

子宫内膜异位症(内异症)是妇科常见病,病因不清。根据Ｓａｍｐｓｏｎ的种植学说,子宫内膜随经血逆流入腹腔导致发病。近年人们开始关注异位种植的“种子”,即内异症患者正常位置子宫内膜(在位内膜)的特性,以探讨其发病机理。现将有关内异症在位内膜的研究综述如下。一、在位内膜变化在内异症发病中的作用(一)细胞凋亡率降低Ｄｉｎｇ等[1]研究发现,正常妇女子宫内膜细胞凋亡在增殖早期和分泌晚期达高峰,内异症患者的在位内膜腺上皮和间质的细胞凋亡率较正常妇女低3～4倍。患者异位内膜细胞凋亡率较相应的在位内膜更低[2],并且异位内膜细胞对…     

孕三烯酮对体外培养异位子宫内膜细胞生长及凋亡的影响

目的　探讨孕三烯酮对体外培养的子宫内膜异位症(内异症)患者的异位子宫内膜细胞(异位内膜细胞)生长及凋亡的影响,及第10号染色体缺失的磷酸酶和张力蛋白同源物(phosphataseandtensionhomologuedeletedonchromosome10,PTEN)基因在异位内膜细胞表达的变化。方法　以0、10. 6和10 .4 mol/L浓度的孕三烯酮,对体外培养的异位内膜细胞分别进行处理;采用四甲基偶氮唑蓝比色法,检测孕三烯酮对异位内膜细胞作用0 ~48h的细胞抑制情况并绘制细胞生长曲线;应用透射电镜观察孕三烯酮对异位内膜细胞作用24h时的细胞超微结构变化;采用流式细胞仪检测异位内膜细胞凋亡率及细胞周期变化;应用PTEN单克隆抗体,经流式细胞仪分析异位内膜细胞PTEN基因的表达。结果　孕三烯酮作用于异位内膜细胞8、16、24、32、40和48h的细胞生长率,在孕三烯酮浓度为10 .6 mol/L时,分别为99 .6%、87 .3%、79 .8%、62 .3%、51. 7%和44. 2%;在10 .4 mol/L时,分别为99 .2%、77 .1%、69. 6%、51 .1%、33 .7%和23 .6%。同一浓度各作用时间比较,差异均有统计学意义(P<0 .05)。孕三烯酮作用24h后,异位内膜细胞发生典型的细胞凋亡形态学改变。浓度为10 6、10 4 mol/L的孕三烯酮作用于异位内膜细胞后,凋亡率分别为1 3%、15 .0%;浓度为0mol/L的孕三烯酮     

米非司酮对离体异位子宫内膜细胞凋亡的影响

目的 :观察培养的异位子宫内膜细胞分组加入米非司酮后的生长情况及形态学变化 ,并检测其凋亡率 ,探讨子宫内膜异位症的药物治疗。方法 :对子宫内膜异位症14例的异位子宫内膜离体组织进行培养 ,在相同的生长期加入一定剂量米非司酮后 ,观察细胞的生长状况并用TUNEL法检测其凋亡率。结果 :加入米非司酮后 ,细胞生长速度减慢 ,其凋亡率较未用药组明显增加 ,差异有显著性 (P <0 .0 5 )。结论 :米非司酮对异位细胞有抑制作用 ,并诱导异位子宫内膜细胞凋亡 ,应用于临床治疗子宫内膜异位症疗效良好。     

异位子宫内膜细胞的凋亡与增殖的研究

目的 探讨异位子宫内膜细胞的凋亡特性及雌二醇（Ｅ２）、孕酮（Ｐ）、米非司酮对体外培养的人异位子宫内膜细胞表皮生长因子受体（ＥＧＦＲ）基因表达的影响。方法 对１５例子宫内膜异位症（内异症）患者的异位内膜组织（异位内膜组）及１１例非子宫内膜异位患者的在位内膜组织（在位内膜组）进行体外细胞培养,采用流式细胞术检测两组细胞的细胞凋亡指数,同时应用原位杂交方法检测细胞中ｂｃｌ－２的表达水平。应用逆转录聚合酶     

子宫内膜增生性疾病患者内膜细胞凋亡的研究

目的 :研究凋亡在子宫内膜增生性疾病中的作用。方法 :用改良原位末端标记技术检测 15例正常月经周期的增生期、分泌期、月经期子宫内膜 ,11例增殖性子宫内膜 ,12例子宫内膜癌 ,以及术前用孕激素治疗的 13例异常增生子宫内膜中的凋亡细胞 ,并计算其凋亡指数 (AI)。结果 :分泌期、月经期子宫内膜、增殖性子宫内膜、子宫内膜癌AI均比正常增生期子宫内膜AI高 (P <0 .0 1)。增殖症患者内膜不典型增生组AI比单纯增生、复杂增生组AI高 (P <0 .0 5 ) ;内膜癌患者低分化组AI比高分化组、中分化组AI高 (P <0 .0 5 )。结论 :细胞凋亡与正常子宫内膜周期性变化有关 ,而在增殖性和癌变子宫内膜中的异常表达可能与子宫内膜的良恶性病变有关     

米非司酮可诱导人子宫内膜细胞的凋亡和坏死

目的:探讨米非司酮对人子宫内膜细胞凋亡和坏死的影响。方法:体外培养人子宫内膜细胞,采用不同浓度米非司酮处理,观察米非司酮对细胞形态的影响,MTT法检测米非司酮对细胞增殖能力的影响,检测米非司酮处理细胞后子宫内膜发育相关基因ESR1和PGR的含量、坏死途径蛋白p-MLKL、RIPK3的表达和凋亡途径蛋白Bax、procaspase3/caspase3的变化。结果:经米非司酮处理后,人子宫内膜细胞细胞形态明显变差,细胞增殖能力明显变弱,且随着米非司酮处理浓度增加,其对细胞生长的毒性作用加强;米非司酮处理的细胞中ESR1和PGR含量明显减少,而坏死途径蛋白p-MLKL、RIPK3表达和凋亡途径蛋白Bax、pro-caspase3/caspase3表达增加。结论:米非司酮可促进人子宫内膜细胞的凋亡和坏死而抑制其细胞增殖,其作用可能与调节ESR1/PGR含量、坏死途径蛋白p-MLKL/RIPK3及凋亡途径蛋白Bax/pro-caspase 3/caspase 3表达有关。     

The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: A randomized study

Purpose: The aim of this study was to examine the influence of endometrial cells on the fertilization rate and early embryonic morphology following routine in vitro fertilization (IVF). Cryopreservation with subsequent thawing allowed the use of autologous somatic cells, thus minimizing the risk of transmission of infective agents. Interpatient variability was eliminated by randomizing oocytes from each cycle into the control or coculture group. Results: Two hundred ninety-four oocytes from 24 IVF cycles (21 patients) were included in the study (145 coculture and 149 control). The normal fertilization rate of control oocytes (56.4%) was not significantly different from that of oocytes cocultured with endometrial cells (61.4%). The mean number of blastomeres in cocultured embryos (3.65) was not significantly different from the number in control embryos (3.46) 2 days after insemination, but the proportion of embryos with minimal or no fragmentation was significantly higher in the coculture group [34/84 (40.5%) vs.17/80 (21.3%);P<0.01]. Conclusions: The inclusion of cryopreserved autologous endometrial cells in routine clinical IVF procedures does not influence fertilization or the early cleavage rate but may reduce the extent of embryo fragmentation during the early cleavage divisions.     

Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development

Problem The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture.Method To determine the ability of mouse cumulus cell coculture in the presence of human follicular fluid to support preimplantation mouse Balb/cJ embryo development in vitro.Results Culture of preimplantation mouse Balb/cJ embryos independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst development or total cell number per blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P<0.07) improved blastocyst development and the total number of cells per blastocyst.Conclusions Cumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro.     

Endometrial secretory proteins enhance early embryo development

Purpose To evaluate the role of endometrial stromal cells and their secretory proteins in early embryo development, two-celled CB6F1 mouse embryos were cultured alone or cocultured with human endometrial stromal cells in various culture conditions.Results The percentage of embryo blastocyst formation, hatching, and outgrowth was significantly greater in (1) coculture with endometrial stromal cells than in a cell-free control when both coculture and control were carried out in protein-free medium or in RPMI 1640 plus 10% fetal calf serum; (2) coculture with hormone (i.e., progesterone plus relaxin)-treated cells than in coculture with hormone-nontreated cells; and (3) media supplemented with isolated endometrial secretory proteins than in media supplemented with BSA (0.35%). Embryo development was not found to be significantly different in coculture and in media supplemented with endometrial secretory protein.Conclusion Our data provides credence to the theory that endometrial stromal cells enhance embryo development by secreting specific proteins that are beneficial to embryo growth in vitro.Presented at the 48th Annual Meeting of the American Fertility Society, New Orleans, Louisiana, October 31–November 5, 1992.     

Potential use of embryo coculture with human in vitro fertilization procedures

Purpose This review was designed to outline potential uses of an embryo co-culture system in human assisted reproduction programs to improve embryo quality and pregnancy rates.Results The various cell types used in embryo co-culture were reviewed in addition to the use of co-culture for both animal and human embryos. Co-culture provides a method to enhance embryo development in an inadequate in vitro environment without compromising embryo quality. Human IVF laboratories have used various types of helper cells to improve rate of development, reduce cell fragmentation rate and in some instances increase pregnancy and implantation rates.Conclusion In conjuction with several assisted reproduction procedures such as IVF, microsurgical fertilization, cryopreservation and genetic evaluation, co-culture may increase the number of viable embryos for replacement and improve pregnancy rates.Presented at the First Asian Symposium on Micromanipulation and Co-culture, April 1, 1993, Singapore.     

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells

Purpose The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared.Results The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 ± 25.9; fresh coculture group, 108.4 ± 25.1) than the control group (87.7 ± 31.9).Conclusion The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.     

Optimizing tubal epithelial cell growth promotes mouse embryo hatching in coculture

Purpose: This study investigates the relationship between human tubal epithelial cell growth characteristics and mouse embryonic development to determine which cellular requirements should be preferentially provided in a coculture system. Methods: Cell growth and viability were assessed for 5 days in -minimal essential medium or human tubal fluid supplemented with 10% human serum or 10% synthetic serum. Two-cell mouse embryo development to blastocyst and hatching blastocyst stages was also assessed with or without coculture. Results: Both epithelial cell growth and embryo development were dependent on serum supplementation with better cell viability and growth rates in human serum and better blastocyst development in synthetic serum. The highest proportion of hatching blastocysts was found in -minimal essential medium and human serum with coculture. Conclusions: Culture conditions which improve tubal epithelial cell growth also improve the hatching rate of mouse embryos in coculture. This indicates that by meeting the metabolic and nutritional demands for epithelial cell growth, the beneficial effects of coculture on embryo development may be optimized.     

Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells

Purpose Although the hatching of embryos is an important phenomenon, the mechanism of hatching remains controversial. Therefore, we attempted to develop a new coculture system with human placental cells to investigate the hatching of mouse embryos.Results In our new system there was no difference in development from the two-cell stage to blastocysts between embryos cultured with a T6 medium and embryos cocultured with human placental cells at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ cells/ml. However, the hatching-rate cell number increased significantly in embryos cocultured with placental cells compared to embryos cultured without placental cells. [³H]Thymidine uptake did not show any significant difference from the beginning of in vitro culture to the hatching stage between the coculture group and the control group. Nevertheless, the [³H]uridine uptake was significantly different in the two groups, measuring 2167 ± 532 cpm/10 embryos in the coculture group and 804 ± 86 cpm/10 embryos in the control group at 114 hr after human chorionic gonadotropin injection (P < 0.01).Conclusion These results therefore seem to indicate that the hatching of blastocysts depends on the protein synthesis of the embryos and not on DNA duplications.     

hCG对卵泡及子宫内膜发育作用的研究进展

人绒毛膜促性腺激素(hCG)可以改善内膜容受性(ER)、延长种植窗口期,有利于胚胎着床,还可以调节子宫内膜的免疫耐受性,从而提高种植率;在卵泡早期或者中晚期添加低剂量的h CG可以提高卵子以及胚胎质量,从而获得更高的妊娠率。     

冻融胚胎移植周期无创性内膜刺激胚胎移植策略的临床疗效探究

目的探讨无创性内膜刺激胚胎移植(stimulation of endometrium embryo transfer,SEET)技术在冻融胚胎移植(frozen-thawed embryo transfer,FET)周期中对妊娠结局的影响。方法回顾性分析2016年3月—2017年2月在本中心进行体外受精-胚胎移植(IVF-ET)并首次行单囊胚FET的不孕症患者的临床资料,按移植方式分组:A组(实验组)57个周期,FET时采用SEET技术;B组(对照组)56个周期,FET时采用传统囊胚移植技术。结果 A组采用SEET技术后的胚胎种植率(64.9%)和临床妊娠率(64.9%)显著高于B组(44.6%,44.6%)。结论 SEET技术可以显著提高临床妊娠率,为改善IVF结局提供了一种新的移植策略。