Comparison of intracytoplasmic sperm injection with testicular spermatozoa success in infertile men with obstructive and non-obstructive azoospermia; a retrospective analysis

The aim of this study was to compare the outcome of intracytoplasmic sperm injection (ICSI) and embryo transfer between couples with infertility due to male non-obstructive azoospermia (NOA) and obstructive azoospermia (OA). A retrospective analysis of 234 couples with azoospermia who were treated by ICSI and embryo transfer between January 2007 and October 2010 was performed. There were 61 couples in NOA group and 173 couples in OA group. Fertilization rates, pregnancy and clinical pregnancy rates were the main outcome measures. The number of retrieved mature oocytes, injected oocytes, metaphase II (MII) oocytes, two distinct pronuclei oocytes, cleavage embryos and embryos transferred was not significantly different between the groups. The fertilization rate was significantly lower in NOA group when compared to OA group (56.2 vs. 66.7%, respectively; p?=?0.013) and the pregnancy rate was significantly lower in NOA group than OA group (36.1 vs. 50.9%, respectively; p?=?0.046). The clinical pregnancy rates were not statistically different between the patients with NOA and OA azoospermia groups (24.6 vs. 36.4%, respectively; p?=?0.09). This study suggests that ICSI and embryo transfer together with testicular sperm extraction results in statistically significant lower fertilization and pregnancy rates in men with NOA when compared to men with OA.     

Differential induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand in human ovarian carcinoma cells

OBJECTIVES: In this study, we examine the sensitivity of a panel of ovarian carcinoma cells, which includes four primary ovarian cancer cell samples, and four normal ovarian epithelium samples to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We also examine the intracellular regulation of TRAIL-mediated apoptosis. METHODS: The sensitivity to TRAIL was determined by short-term survival assays on seven ovarian carcinoma cell lines, four primary samples of ovarian cancer, and four normal ovarian epithelium samples. We assessed the activation of the apoptotic pathway in TRAIL-resistant and -sensitive tumor cells. The expression of TRAIL receptors was determined by flow cytometry. The protein expression of FADD, XIAP, caspase-8, caspase-3, BAX, and c-FLIP were determined by immunoblot analyses. RESULTS: We show that ovarian cancer cells display variable sensitivity to TRAIL-induced apoptosis although most cell lines have similar sensitivity to cisplatin. Normal ovarian epithelium samples were mostly sensitive to TRAIL. In sensitive cells, TRAIL induced caspase-8-dependent apoptosis, which subsequently led to activation of caspase-3. Both sensitive and resistant cells expressed caspase-8, caspase-3, FADD, XIAP, and c-FLIP at similar levels. A significant enhancement in cell death was observed in TRAIL-resistant cells when c-FLIP(L) levels were downregulated by RNA interference. CONCLUSIONS: These data suggest that sensitivity to TRAIL and chemotherapy does not necessarily correlate in human ovarian cancer cells. Cancerous cells isolated from patients with ovarian cancer show variable sensitivity to TRAIL but most normal ovarian epithelial cells are sensitive. In human ovarian cancer cells, c-FLIP(L) may participate to the regulation of the TRAIL signaling cascade.     

Survivin和Caspase-3在结肠癌和结肠腺瘤组织中的表达及意义

为探讨Survivinfp Caspase-3在结肠癌和结肠腺瘤组织中的表达及意义,应用免疫组织化学染色法对42例结肠癌正常黏膜组织、38例结肠腺瘤组织进行Survivin和Caspase-3检测。结果显示,结肠癌组织中Survivin阳性率明显高于结肠腺瘤和结肠正常黏膜组织(P<0.05)结肠癌组织中Caspase-3阳性率明显低于结肠腺瘤组织(P<0.05)。Survivine-3在结肠癌组织中的表达呈负相关(r--0.8535,P=0.000)。结果表明,Survivin可能通过抑制结肠癌细胞凋亡,对肠癌的发生发展起重要作用;在结肠癌的癌变过程Survivin的作用可能是通过抑制Caspase-3介导的细胞凋亡实现的。     

纳秒级脉冲电场对SKOV3细胞的凋亡诱导作用及对凋亡相关蛋白Bcl-2和Bax表达的影响

目的:探讨纳秒级脉冲电场对SKOV3细胞的凋亡诱导作用及对凋亡相关蛋白Bcl-2/Bax表达的影响。方法:以场强为50kV/cm,脉宽100ns,10、30、60或80个脉冲电场处理SKOV3细胞,以未经处理的SKOV3细胞为对照组,Annexin V/PI双染流式细胞仪检测早期凋亡;处理后6h,琼脂糖凝胶电脉法检测晚期凋亡;以50kV/cm,100ns,30个脉冲电场处理细胞,分别孵育0h,2h,6h,12h后提取总蛋白,Western blot法检测caspase-3活性片段、Bax、Bcl-2表达量的改变。结果:与对照组相比,纳秒级陡脉冲处理组细胞出现明显的磷脂酰丝氨酸(PS)外翻(P0.05)且细胞膜保持完整,即Annexin V(+)/PI(-);凝胶电泳检测显示,处理组DNA呈明显的阶梯状分布;Western blot检测示,与对照组相比,处理组细胞内caspase-3活性片段、促凋亡蛋白Bax表达量显著增加(P0.05),凋亡抑制蛋白Bcl-2表达量略降低,Bcl-2/Bax显著降低(P0.05)。结论:纳秒级脉冲电场可通过caspase-3依赖凋亡途径,降低Bcl-2/Bax比值诱导SKOV3细胞凋亡。     

早产孕妇蜕膜细胞凋亡、周期变化及Bcl-2、Caspase-3和p21蛋白的表达研究

目的 探讨早产孕妇蜕膜细胞的凋亡、细胞周期及Bcl-2、Caspase-3、p21蛋白的表达情况.方法 选取90例孕产妇入组,分为早产组(n=30)、足月顺产组(n=30)和足月剖宫产组(n=30).比较三组孕产妇蜕膜细胞的细胞周期、凋亡情况及相关蛋白Bcl-2、Caspase-3、p21的表达.结果 早产组患者蜕膜细...     

Caspase-3 gene expression in human luteinized granulosa cells is inversely correlated with the number of oocytes retrieved after controlled ovarian stimulation

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman’s r?= ?0.308), the number of collected oocytes (r?= ?0.451), the number of mature oocytes (r?= ?0.526), the number of fertilized oocytes (r?= ?0.439) and the number of viable embryos (r?= ?0.443, all statistically significant at p?相似文献   

Apoptosis and active caspase-3 expression in human granulosa cells

OBJECTIVE: To document the expression of activated forms of caspase-3 in human granulosa cells. DESIGN: Laboratory study. SETTING: In vitro fertilization (IVF) laboratory of the Split University Hospital and laboratory of the Department of Anatomy, Histology, and Embryology. PATIENT(S): Ovarian tissues were obtained from women undergoing hysterectomy/ovariectomy for benign conditions and human granulosa cells were obtained from women undergoing oocyte retrieval for IVF. INTERVENTION(S): Immunostaining of tissue sections and cell smears using antibody to active caspase-3 and terminal deoxynucleotidyl transferase (TdT) assay (TUNEL) for detection of internucleosomal DNA fragmentation. MAIN OUTCOME MEASURE(S): Microscopic evaluation to assess the presence and cellular co-localization of active caspase-3 and TUNEL-positive cells. RESULT(S): In human ovarian tissue, no apoptosis was observed in primordial and primary follicles. Apoptosis in granulosa cells was detected only in atretic antral follicles. Granulosa cells classified as apoptotic on the basis of their morphologic features contained a single condensed nucleus, multiple nuclear fragments, or apoptotic bodies. All apoptotic granulosa cells expressed active caspase-3, but only few contained fragmented DNA detected with the TUNEL method. The expression of active caspase-3 was also demonstrated in human granulosa cells of preovulatory follicles obtained from patients undergoing IVF. CONCLUSION(S): Caspase-3 dependent apoptosis occurs in human granulosa cells and activates when follicles begin to leave the resting pool. After initial formation of the antrum, activation of caspase-3 is a normal physiologic process of the follicle during atresia and luteinization. Higher numbers of granulosa cells positive with caspase-3 than cells positive with TUNEL suggest an earlier activation of caspase-3 compared with the DNA fragmentation detected by TUNEL assay and also a longer detection period of caspase-3 than DNA fragmentation in apoptotic granulosa cells.     

Divergent regulation of angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor by hypoxia and female sex steroids in human endometrial stromal cells

Objective

To investigate whether hypoxia or the female sex steroids exert direct effects on angiopoietin-1 (ANGPT1), ANGPT2, and vascular endothelial growth factor (VEGF) in human endometrial stromal cells (ESCs) to clarify the regulatory function of these local angiogenic factors in the endometrium.

Study design

Human endometrial tissues were obtained from 18 patients aged 34–47 years undergoing hysterectomy for benign reasons. ESCs were cultured under hypoxic condition or treated with 17β-estradiol (E) and/or medroxyprogesterone acetate (MPA). The mRNA levels and production of ANGPT1, ANGPT2, and VEGF were assessed by real-time RT-PCR and ELISA, respectively. Analysis of hypoxia-inducible factor 1α (HIF-1α) and estrogen receptor α (ERα) protein levels were evaluated by Western blot analysis.

Result(s)

Hypoxia reduced the mRNA expression and protein production of ANGPT-1 in ESCs, whereas those of ANGPT2 were unaffected, resulting in an increase of the ANGPT2/ANGPT1 ratio. Hypoxia induced mRNA expression and protein production of VEGF. E simultaneously induced VEGF production and suppressed ANGPT1 production, resulting in an increase of the ANGPT2/ANGPT1 ratio. MPA or E + MPA reduced ANGPT2 production and sustained the levels of ANGPT1, resulting in a decrease of the ANGPT2/ANGPT1 ratio. With regard to the interaction of E and hypoxia, E did not affect the regulation of angiogenic factors, HIF-1α, and ERα under hypoxic conditions.

Conclusions

Hypoxia and female sex hormones independently regulate the ANGPT2/ANGPT1 ratio and VEGF expression in human ESCs. These results may indicate a potential mechanism for hypoxia or female sex steroids influencing angiogenesis in the human endometrium.     

重度子痫前期患者胎盘组织中Survivin和Caspase-3表达

目的探讨重度子痫前期患者胎盘组织中Survivin和Caspase-3的表达,以及其与早发型重度子痫前期的发生,发展关系。方法采用免疫组织化学染色方法检测20例正常妊娠孕妇(对照组)、40例重度子痫前期患者(重度子痫前期组,其中早发型20例,晚发型20例)的胎盘组织进行Survivin和Caspase-3的定位,并用计算机图像分析系统进行定量分析比较。结果重度子痫前期组与对照组比较,Survivin在胎盘滋养层细胞的细胞浆及部分细胞核中的表达低于对照组,差异有统计学意义(P0.05),Caspase-3在胎盘滋养层细胞的细胞膜、细胞浆中的表达高于对照组,差异有统计学意义(P0.05)。且两者呈负相关关系(r=-0.84,P=0.0001)。早发型重度子痫前期与晚发型重度子痫前期比较,Survivin显著降低,而Caspase-3表达增加。结论Survivin的表达降低,Caspase-3的表达升高可能与重度子痫前期的发生有关。     

磷脂酰肌醇3激酶通路在胰岛素调节子宫内膜癌细胞增殖凋亡中的作用

目的 探讨磷脂酰肌醇3激酶(PI3K)信号通路在胰岛素调节子宫内膜癌细胞生长中的作用.方法 2006年5月至2006年10月于中国医学科学院血液病学研究所临床药物室完成试验,即将子宫内膜癌Ishikawa3-H-12细胞进行无血清饥饿培养,分成不同处理组,即空白对照组、胰岛素刺激组(Ins组)、PI3K抑制剂--LY294002预处理后再以胰岛素刺激组(LY Ins组).以四甲基偶氮唑蓝(MTT)试验和流式细胞仪观察细胞增殖和凋亡情况.不同处理组分别培养24h、48h和72h,观察各组570nm波长吸光度(OD570nm).结果 Ins组细胞OD570nm值高于空白对照组(P＜0.01);LY Ins组细胞OD570nm低于Ins组(P＜0.01),LY294002拮抗了胰岛素对内膜癌细胞的增殖促进作用.流式细胞仪检测凋亡发现Ins组细胞凋亡率低于空白对照组[(2.02±0.39)%对(14.45±2.70)%,P＜0.01];LY Ins组细胞凋亡率高于Ins组[(18.73±2.11)%对(2.02±0.39)%,P＜0.01].结论 PI3K信号通路在胰岛素引起的促内膜癌细胞增殖、抑制凋亡中起重要作用,胰岛素对子宫内膜癌细胞增殖、凋亡的调节作用是PI3K依赖性的.     

沉默PI3K基因对E2刺激后子宫内膜癌细胞增殖和凋亡的影响

目的:探讨沉默PI3K基因对雌激素刺激下的子宫内膜癌细胞增殖、迁移、凋亡的影响。方法:培养子宫内膜癌Ishikawa细胞,构建LV-PI3K-RNAi慢病毒载体转染Ishikawa细胞。实验分组Control组未经任何处理的Ishikawa细胞;Ishikawa组经E₂刺激的Ishikawa细胞;NC组经E₂刺激并转染阴性对照慢病毒的Ishikawa细胞;PI3Ki组经E₂刺激并转染LV-PI3K-RNAi慢病毒的Ishikawa细胞。Real-time PCR、Western blot法检测各组VEGF、bFGF、PI3K mRNA及蛋白表达水平,MTT法、流式细胞仪分别检测细胞增殖能力及凋亡的情况。结果:与Ishikawa组、NC组比较,PI3Ki组的VEGF、bFGF、PI3K mRNA及蛋白表达水平明显降低,细胞增殖能力显著降低,凋亡率增高,差异均有统计学意义(P<0.05)。结论:PI3K基因低表达可干扰E₂在基因、蛋白水平激活Ishikawa细胞产生VEGF、bFGF,从而下调E₂对子宫内膜癌细胞增殖和抑制凋亡的影响。     

siRNA LSINCT5联合顺铂对卵巢癌SKOV3细胞增殖和凋亡的影响

目的:探讨沉默LSINCT5联合顺铂对人卵巢癌SKOV3细胞增殖及凋亡的影响。方法:选取SKOV3细胞株,采用小干扰技术将LSINCT5基因片段转入卵巢癌SKOV3细胞中,同时联合顺铂作用于卵巢癌细胞。按不同处理因素将SKOV3细胞分为5组:对照组、NC-siRNA组、顺铂组、LSINCT5-siRNA+顺铂组和LSINCT5-siRNA组。CCK-8检测转染后SKOV3细胞对顺铂的敏感性。显微镜下观察细胞的生长状况及形态变化。Western blot和RT-PCR法分别检测与凋亡相关的Caspase 3蛋白和基因表达水平。结果:CCK-8显示,3μg/ml顺铂作用于卵巢癌细胞的抑制率较为适中,故作为后续实验的处理浓度。沉默LSINCT5能显著提高卵巢癌细胞对顺铂的敏感性。LSINCT5-siRNA+顺铂组中Caspase 3蛋白和mRNA相对表达量均明显高于其他各组(P0.05)。结论:沉默LSINCT5提高了卵巢癌细胞对顺铂的敏感性。沉默LSINCT5联合顺铂能明显抑制卵巢癌细胞的增殖,促进卵巢癌细胞凋亡。     

HER2蛋白在上皮性卵巢癌中的表达及曲妥珠单抗对卵巢癌SKOV3细胞的影响

目的:探索HER2蛋白在上皮性卵巢癌组织中的表达及新型HER2靶向治疗药物曲妥珠单抗(赫赛汀)对卵巢癌SKOV3细胞的影响。方法:1采用免疫组化SP法、实时定量聚合酶链式反应(qRT-PCR)法检测HER2蛋白在58例上皮性卵巢癌组织(恶性组),20例卵巢良性上皮性肿瘤组织(良性组)、10例正常卵巢组织(正常组)中的表达情况;2体外培养人上皮性卵巢癌SKOV3细胞,采用四甲基偶氮唑盐比色法(MTT)、流式细胞术(FCM)检测不同浓度赫赛汀对卵巢癌SKOV3细胞增殖、凋亡的影响;采用蛋白印迹法检测SKOV3细胞早期相关凋亡蛋白Caspase-3的表达水平。结果:1HER2蛋白在恶性组的阳性表达率显著高于良性组及正常组(P0.05),且随临床分期及肿瘤组织学分级的升高而增加(P0.05),有淋巴结转移的阳性表达率高于无转移者(P0.05)。2随赫赛汀浓度的升高,卵巢癌SKOV3细胞G0、G1期比例明显增高(P0.05),而G2、M期和S期的细胞比例明显降低(P0.05),不同浓度赫赛汀组与对照组比较,差异有统计学意义(P0.05);细胞凋亡率随赫赛汀的药物浓度增加而逐渐增高,赫赛汀组与对照组比较及不同浓度赫赛汀组间细胞凋亡率比较,差异有统计学意义(P0.05)。3各浓度赫赛汀组Caspase-3蛋白相对表达量比较,差异有统计学意义(P0.01);各浓度组与对照组比较,差异有统计学意义(P0.05)。结论:HER2蛋白在上皮性卵巢癌中呈高度表达,且表达与肿瘤分期、细胞分化程度及淋巴结转移相关。周期阻滞、凋亡诱导可能是赫赛汀抗肿瘤机制之一。     

Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer

Objectives

We have examined the role of VEGFC/VEGFR3 signaling in lymph node metastasis and growth of orthotopic human ME180 and SiHa cervical xenograft models following exposure to hypoxia. Our previous studies showed that growth of these tumors under conditions of cyclic hypoxia increased nodal metastasis.

Methods

Mice bearing orthotopic tumors were subjected to cyclic hypoxia at 7% O₂/air 10 min cycles 4 h/day/2 weeks. Knockdown of vegfc was carried out by shRNA and inhibition of VEGFR3 was conducted by blocking antibodies for the mouse and human proteins. VEGFR3 protein expression was detected by Western blotting. Immunohistochemical staining was used to assess CA9, CD31, LYVE1 and Ki67 labeling. Gene expression was determined by real time PCR.

Results

Knock down of vegfc or inhibition of VEGFR3 with blocking antibody reduced metastases under normoxic and cyclic hypoxia conditions. A reduction in lymphatics and blood vessel formation and a decrease in tumor cell proliferation was observed following vegfc knockdown and VEGFR3 inhibition. VEGFR3 expression was upregulated at the mRNA and protein levels following hypoxia.

Conclusions

Collectively, our results indicate that anti-VEGFR3 antibody inhibits vegfc-induced tumor lymphangiogenesis and metastasis and that vegfc knockdown in the tumor cells causes a similar inhibitory effect on lymph node metastasis. These results suggest that the effects of vegfc/VEGFR3 on the progression of tumor cells to form lymph node metastases occur primarily under an hypoxic tumor microenvironment.     

iNOS和caspase 3在胚胎停育绒毛和蜕膜组织中的表达

目的：探讨胚胎停育绒毛组织和蜕膜组织中半胱氨酸天冬氨酸蛋白酶3（caspase 3）和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的表达和临床意义。方法：选择2013年3月-2014年5月就诊于武警后勤学院附属医院妇产科计划生育门诊的30例不明原因早期胚胎停育的患者（病例组）和30例正常早期妊娠妇女（对照组）为研究对象,采用逆转录聚合酶链反应（RT-PCR）方法检测2组绒毛组织和蜕膜组织中caspase 3和iNOS mRNA的表达水平,采用免疫印迹法检测2组绒毛组织和蜕膜组织中caspase 3蛋白的表达水平。结果：①病例组绒毛组织和蜕膜组织中caspase 3 mRNA、总的caspase 3蛋白和活化的caspase 3蛋白的表达水平均高于对照组（P＜0.01）。②病例组绒毛组织和蜕膜组织中iNOS mRNA表达水平低于对照组（P＜0.01）。结论：正常早孕和胚胎停育患者绒毛组织和蜕膜组织中均有iNOS和caspase 3的表达,iNOS和caspase 3可能参与凋亡的增加进而引起胚胎停育的发生。     

芬太尼通过PI3 K/Akt信号通路调控宫颈癌细胞增殖迁移

目的:探究芬太尼对宫颈癌细胞增殖迁移的作用及分子机制.方法:将宫颈癌细胞系C33 A分为对照组和芬太尼组(使用不同浓度芬太尼处理),分别采用CCK-8、Transwell法、流式细胞术检测芬太尼对C33 A细胞增殖、迁移及凋亡的影响.Western blot法检测活化Caspase 3、PARP1含量,MMP-2、MM...