Background Utilizing Raman spectroscopy detection technique for assessing the clinical pregnancy outcomes following in vitro fertilization-embryo transfer (IVF-ET). Methods A total of 76 couples took part in the research. During their infertility treatment, culture medium samples were collected from 76 embryos (40 pregnancy and 36 non-pregnancy) at both the D3 and D5 stages. In total, 747 Raman spectra were recorded from these samples. The statistical analysis results were evaluated using accuracy, precision, recall, F1-score, and Area Under the Curve(AUC) to assess the classification performance of the fusion analysis model. The biochemical analysis results involved comparing the significant differences in spectral signal intensities of embryo metabolites corresponding to different pregnancy outcomes. Results This study established a fusion analysis model based on Raman spectroscopy data for predicting the pregnancy potential of embryos. The combination model employing Hilbert-PCA, spectrogram concatenation fusion, and SVM achieved an accuracy of over 93%. Furthermore, the statistical analysis results indicated significant differences in Raman spectroscopy features obtained from culture medium samples of embryos with varying pregnancy potentials. Conclusions This study utilized non-invasive Raman spectroscopy technology to analyze the metabolic spectra of culture medium across multiple developmental stages. By establishing a fusion analysis model, high-precision prediction of embryo pregnancy potential was achieved. Trial registration: This study, with ethical review number 2021 − 384, was approved by the Institutional Review Board of Yuhuangding Hospital, affiliated with Qingdao University.

This study aimed to examine the performance of the dual antiplatelet therapy (DAPT) score in two retrospective cohorts of post-percutaneous coronary intervention (PCI) patients and to explore whether incorporating additional biomarkers could further improve the predictive power of the DAPT score. In a retrospective derivation cohort of 4,798 PCI patients, the validity of DAPT score for stratifying ischemic/bleeding risks was explored. Then, the association between the baseline status of 54 laboratory test biomarkers and ischemic/bleeding events was revealed while adjusting for the DAPT score. Combinations of individual laboratory test biomarkers that were significantly associated with ischemic/bleeding events were explored to identify the ones that improved discrimination of ischemic and bleeding events when incorporated into DAPT score. Finally, the impact of the combination of biomarkers with DAPT score was validated in an independent retrospective validation cohort of 1,916 PCI patients. Patients with a high DAPT score (DAPT score ≥ 2) had significantly higher risk of ischemic events and significantly lower risk of bleeding than patients with a low DAPT score (DAPT score < 2). Moreover, the addition of aspartate aminotransferase (AST) and red cell distribution width CV (RDW-CV) into the DAPT score further improved discrimination of ischemia and bleeding. Furthermore, the incremental predictive value of AST + RDW-CV maintained with measurements was updated at post-baseline time points. DAPT score successfully stratified the risks of ischemia/bleeding post PCI in the current cohorts. Incorporation of AST + RDW-CV into the DAPT score further improved prediction for both ischemic and bleeding events.

目的 制备高活性EB病毒立即早期蛋白Rta优势表位抗原,并初步评价该抗原在鼻咽癌(nasopharyngeal carcinoma,NPC)诊断中的价值.方法 分析Rta氨基酸序列,选取抗原优势表位Rta(301～440aa),合成目的基因,连接pBVGST-6His载体,构建重组pBV-Rta表达质粒,进行诱导表达获得纯化抗原.采用间接ELISA初步评价优势表位抗原在鼻咽癌诊断中的价值,以纯化的pBV-Rta优势表位抗原为包被抗原,抗人IgA,IgG和IgM为二抗,检测由烟台毓璜顶医院提供的50例经临床病理确诊的NPC患者血清样本和90例健康体检者血清样本.结果 获得了高效原核表达的pBV-Rta优势表位抗原(301～440aa),经过小样本验证,确定pBV-Rta具有良好的抗原活性和特异度.放大样本进行检测,基于Rta优势表位抗原的Rta-IgG间接ELISA方法检测灵敏度和特异度分别为73.08％和95.79％.Rta-IgG检测的AUC值为0.860.结论 Rta优势表位抗原pBV-Rta是优选的可以用于Rta-IgG检测的抗原,利用该抗原所建立的间接ELISA检测方法可以很好地区分鼻咽癌患者和健康对照.

The adipokines are secreted by adipose tissue,which maintain the energy steady state,regulate the balance of glucose and lipid metabolic,and play an important role in the occurrence and development of coronary heart disease,directly or indirectly involved in the pathophysiological process of atherosclerosis.With the rapid development of molecular biology and the human genome project,the pathogenesis of coronary heart disease is clarified from the gene level,and now it is becoming a hot spot in the medical research field.Exploration of the distribution of single nucleotide polymorphisms of adipokines genes including adiponectin,resistin,visfatin and serum amyloid A and the relationship between these single nucleotide polymorphisms and coronary atherosclerosis for populations in different ethnic and geographic groups,is of great significance to improve the prevention and treatment of coronary heart disease.Therefore,the genotype detections for adipocytokines and the analysis of the correlation between gene single nucleotide polymorphisms with genetic susceptibility of coronary atherosclerosis actively in future,may provide new measures to prevent,diagnose,and treat coronary heart disease.

MicroRNAs(miRNAs)are a kind of endogenous non-coding small RNA molecules,with stable structure in the blood circulation and slow degradation,which combine with target mRNA,leading to the target mRNA degradation or translation inhibition to regulate the protein expression level after transcription in most eukaryotic cells.Studies have shown that miRNAs take important roles in embryonic morphogenesis,cell proliferation,differentiation and apoptosis,participating in a series of important processes in life,including fetal development and pathological pregnancy outcome.miRNAs expres-sion level changes are closely related to the pathological pregnancy outcome of the embryonic development and implantation, abnormal fetal growth,recurrent abortion,ectopic pregnancy,and preeclampsia.Placental circulation miRNAs are related to pregnancy status,so screening of different miRNAs can be used for monitoring and diagnosis of pregnancy.Screening and identification of differential expression of miRNAs in peripheral blood,are expected to be potential new molecular markers for the pathological state of pregnancy, which have potential application value as noninvasive screening and diagnosis molecular markers.

Objective: To identify the quantitative profiling of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) in cumulus cells (CCs) and to evaluate their roles in the early embryo development of polycystic ovary syndrome (PCOS) patients, in part, with obesity.Methods: Fifty-five subjects were divided into two groups according to the body mass index. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. Real-time PCR and western blot were performed to identify adiponectin and its receptors in CCs.Results: Adiponectin and receptors were ubiquitously expressed in CCs of PCOS and non-PCOS patients. The level of AdipoR2 in CCs from the oocytes yielding blastocyst after 5/6 days in vitro culture was markedly higher than in those from oocytes could not develop to blastocyst stage after Day 6, for non-obese or obese PCOS patients (0.16470.0161 versus 0.07830.0385, 0.19480.0307 versus 0.10570.0236, respectively, p<0.05). In addition, only in patients with PCOS and concurrent obesity the AdipoR1 in CCs was considerably increased in CC-B+ compared with CC-B- subgroup (0.51620.0371 versus 0.2448 +/- 0.0333, p<0.01).Conclusion: The development of early embryo was associated with the up-regulation of AdipoR1 and AdipoR2 in PCOS patients. Our results suggested that adiponectin could positively modulate embryo development in humans. Further investigations should be carried out to unlock the crucial role that adiponectin plays in embryo development.

This study aims to investigate the protective effect of apigenin on the development of experimental autoimmune myocarditis (EAM) and the underlying mechanisms. An EAM model was induced in BALB/c mice by the injection of porcine cardiac myosin. Apigenin was orally administered from day 1 to 21. The severity of myocarditis was assessed by determination of heart weight/body weight ratio (HW/BW) and histopathological evaluation. Echocardiography was conducted to evaluate the cardiac function and heart structure. Antigen-specific T cell proliferation responses to cardiac myosin were evaluated by the lymphocyte proliferation assay. ELISA was used to determine serum levels of type 1 helper (Th1) and Th2 cytokines. Apigenin treatment significantly decreased HW/BW. Histopathologic analysis showed that the infiltration of inflammatory cells was reduced significantly by apigenin treatment. Meanwhile, apigenin administration effectively ameliorated autoimmune myocarditis-induced cardiac hypertrophy and cardiac dysfunction as well as inhibited lymphocyte proliferation in mice immunized with myosin. Furthermore, Th1 cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-2 (IL-2) were significantly downregulated, while Th2 cytokines IL-4 and IL-10 were markedly upregulated. The results indicated that apigenin can alleviate EAM due to its immunomodulatory reactions in modification of helper T cell balance.

To investigate the effect of apigenin on scrotal heat-induced damage in mice, 8-week-old ICR mice were divided into five groups: (I) control group; (II) heat-treat (H) group; (III) H + apigenin 10 mg/kg; (IV) H + apigenin 20 mg/kg; (V) H + apigenin 50 mg/kg. Serum testosterone and inhibin B levels by immunoassays were evaluated. The related oxidative damage markers were determined by kit and histopathological examination was also performed. The results showed that the administration of apigenin was shown a significant effect on the recovery of testis function. When compared with the H group, the H + apigenin groups showed a significant increase in testosterone and inhibin B levels. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity increased after treated by apigenin, but malondialdehyde (MDA) reduced. Histopathological examination further verified the effects of apigenin on scrotal heat-induced damage mice. In conclusion, the results of the present study indicate for the first time the ability of apigenin in attenuation of heat-induced testicular damage, apigenin therapy may be useful as a suppressor of degenerative effects following testicular hyperthermia.

Cerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved neurotrophic factor (NTF) which can protect and repairs dopamine neurons in animal models of Parkinson's disease. The crystal structure of CDNF is dominated by eight α-helices and the 3-dimensional structure of CDNF consists of an N-terminal saposin-like domain and a C-terminal SAP-domain. In particular, the C-terminal domain contains two critical motifs, a CXXC motif and a putative ER retention signal sequences (KTEL) at the C-terminus, which indicate that CDNF may be involved in endoplasmic reticulum (ER) stress. In this study, we found that disrupting helix-7 in the C-terminal could significantly increase CDNF secretion. Moreover, we identified the 149aa-154aa is the key amino acids in helix-7. In all, these findings suggest that helix-7 in the C-terminal is important for CDNF secretion, which maybe potential affect CDNF function in ER stress.

Objective To investigate the expression,localization and function of Ribonuclease 9 (RNase9) in the male reproductive system,so as to provide a theoretical basis for the diagnosis and treatment of infertility caused by abnormal RNase9 expression. Methods Bioinformatic techniques were used to analyze the structure and thereby predict the function of human RNase9. Recombinant human RNase9 protein was obtained using a prokaryotic expression vector constructed by recombinant DNA technology. RT?PCR was performed to evaluate the difference in RNase9 protein expression in various tissues. The RNase9 protein expression in epididymis was compared among elderly[aged(76.3±8.1)years],adults[aged(31.2± 5.6)years]and fetuses [(0.8±0.2)years]. Fluorescence immunohistochemistry was used to localize RNase9 proteins in the testis,epididymis and sperm. Ribonuclease activity of RNase9 was measured with yeast tRNA substrate assay. Sperm suspensions were added with pre?immune serum(Group A)or mouse anti?RNase9 antiserum(Group B),and then subjected to antibody?blocked sperm motility assay to determine the effect of RNase9 protein on indicators of sperm motility[mean curvilinear velocity(VCL),mean straight line velocity (VSL),mean path velocity(VAP)]. From 8 to 12 week?old Golden hamsters intramuscularly injected with progesterone and human chorionic gonadotropin,ova were harvested and incubated with the sperms from Groups A and B(the sperms in Group A had been blocked with pre?immune serum and those in Group B with anti?RNase9 antiserum for 5 h;there were 60 ova used in group A and 62 in Group B). The effect of RNase9 on ovum penetration ability of the anti?body blocked sperms was evaluated by hamster ovum penetration assay. Results Bioinformatic analysis indicated that RNase9 gene is a new member of RNase A superfamily,with a 23?aminoacid signal peptide sequence at the N?terminal,a relative molecular weight of about 20 000 and an isoelectric point(PI)of 5. 92. RNase9 was shown to contain five N?glycosylation sites, two casein kinaseⅡphosphorylation sites,one tyrosine kinase phosphorylation site,one NDUFC2 site and two labeling sites of pancreatic?ribonuclease functional domain. RNase9 protein was ubiquitously expressed in human epididymis,testis,heart,lung,liver,spleen,kidney,and stomach tissues,whereas the expression level was higher in the epididymis. Expression of RNase9 in epididymis was significantly higher in adults than that in the epididymis of fetuses and elderly(all P<0.05). RNase9 protein specifically bound to the equatorial neck region of sperms. Prokaryotic expression yielded to obtain recombinant RNase9 protein and the corresponding antibody with high purity and biological activity. Yeast tRNA substrate assay showed that the RNase9 protein was deficient of ribonuclease activity (0.111 ± 0.007). Antibody blocking was not found to affect the sperm motility of ovum?penetrating ability,with no statistically significant differences in VAP,VSL,VCL and fertilization rate between the two groups(all P>0.05). Conclusion Expression and localization of RNase9 protein in the epididymis and sperm suggest that the protein may play an important role in sperm maturation and development,but may not affect the motility and ovum?penetrating ability of mature sperms rats.

目的：探索Per2在CCAAT增强子结合蛋白α（C／EBPα）参与调控的慢性粒细胞白血病（CML）发生、发展中的作用。方法通过脂质体介导将真核表达载体pGenesil-3-SiPer2和空载体pGenesil-3-HK转染到pEGFP-C／EBPα-K562细胞，利用新霉素筛选稳定干扰Per2的pEGFP-C／EBPα-K562单克隆细胞株。利用 RT-PCR 和 Western blot 分别检测转染组、空载组、对照组细胞p53、c-myc 、cyclinB1的mRNA及蛋白表达水平的改变。结果成功构建稳定干扰Per2表达的pEGFP-C／EBPα-K562细胞株。与对照组和空载组细胞相比，转染组pGenesil-3-SiPer2-K562细胞的p53 mRNA及蛋白表达水平明显降低，差异有统计学意义（P＜0．01），而cyclinB1和c-myc基因的mRNA及蛋白表达水平则显著升高，差异有统计学意义（P＜0．01）。结论 Per2基因在C／EBPα参与调控的CML发生发展中起重要作用，该通路的发现对于研究CML的发生及调控机制具有重要意义。

目的 构建稳定表达转录因子增强子结合蛋白α(CCAAT-enhancer-binding protein α,C/EBPα)基因的白血病髓性细胞系.方法 利用DNA重组技术将C/EBPα基因插入到慢病毒表达载体PLVX-EGFP-3FLAG-Puro中,构建重组慢病毒质粒PLVX-C/EBPα-3FLAG-Puro,与慢病毒包装辅助质粒plp1、plp2、plp/VSVG(1∶1∶1)共转染293细胞,包装出慢病毒,实时荧光定量PCR法检测病毒滴度;收集病毒,分别以10、20 MOI感染K562细胞,G418筛选阳性克隆,利用有限稀释法筛选稳定转染的单克隆细胞株,并采用Western blot法检测C/EBPα蛋白的表达.结果 重组慢病毒质粒PLVX-C/EBPα-3FLAG-Puro经菌落PCR及测序鉴定证明构建正确;包装后的慢病毒pSB957的病毒滴度为5×106 TU/ml;pSB957-K562细胞中C/EBPα蛋白的表达水平较高,且20和10MOI pSB957感染的K562细胞中,C/EBPα蛋白的表达水平无明显差异.结论 成功构建了pSB957-K562白血病细胞株,为进一步研究C/EBPα在白血病发生发展中的作用提供了良好的细胞模型.

目的 研究授精前剥除部分卵丘细胞的卵母细胞的受精、胚胎发育和临床结局. 方法 授精前用机械法剥除卵母细胞外包裹的部分卵丘细胞,每个卵母细胞采用微滴法单独受精(A组).对照组(B组)采用常规体外受精-胚胎移植(IVF-ET)受精方式进行.比较两组受精率(2PN、1PN和3PN)、卵裂率(2PN、1PN和3PN)、优胚率、临床妊娠率、种植率和流产率. 结果 A组和B组的受精率分别是76.51％和73.74％(2PN受精率分别是60.64％,61.62％;1PN受精率分别是1.61％,0.86％；3PN受精率分别是10.44％,9.69％)、卵裂率分别是98.95％和99.17％(2PN卵裂率分别是97.02,98.01％;1PN卵裂率分别是100％,85.71％；3PN卵裂率分别是100％,100％)、优质胚胎率分别是60.75％,62.68％、临床妊娠率分别是63.89％,61.76％、种植率分别是48.00％,38.78％、流产率分别是9.09％,11.90％.所有指标均无统计学意义. 结论 卵母细胞授精前剥除部分卵丘细胞不会影响IVF-ET的临床结局,为利用卵丘细胞进行非侵入性种植前遗传学诊断提供临床依据.

Objective To analyze the aneuploidy of single blastomeres from nonpronuclear embryos on cleavage-stage in in vitro fertilization and embryo transfer (IVF-ET) cycle by microarray comparative genomic hybridization (microarray-CGH). Methods The four non-pronuclei embryo were achieved in one IVF cycle, and microarray-CGH was applied to analyze the aneuploidy of single cells biopsied from the four cleavage-stage embryos on day 3 after insemination. Results The four blastomere had non euploid variation in 24 chromosomes. Out of them, one had X monomer in sex chromosome, and the other three had normal XY diploid in sex chromosome. The four embryos had autosomal mutations, such as gene deletion. Conclusions The aneuploidy analysis by microarray-CGH shows that the blastomeres from nonpronuclear embryos on cleavage-stage in IVF cycle are aneuploid,and nonpronuclear embryos have the risk of abnormal sex chromosome and autosome chromosomes. Key words: in vitro fertilization and embryo transfer; microarray comparative genomic hybridization; non-pronuclei; blastomere

<正>患者1女性,28岁。因"结婚5年未采取避孕措施而未孕",于2006-01-15在本中心就诊。病史采集:婚后第2年,孕龄2个月时自然流产1次(原因不详);婚后第3年宫外孕1次(保守治疗)。子宫输卵管X射线造影提示,双侧输卵管弥散局限。男方精液检查提示,少弱畸形精子症。本中心予促排卵指

目的 探讨精索静脉曲张(VC)患者静脉曲张程度对卵泡刺激素(FSH)、黄体生成素(LH)、睾酮(T)和泌乳素(PRL)4种生殖激素水平的影响.方 法采用电化学发光法,分别对93例不同精索静脉曲张程度的患者(分为3组,Ⅰ度组31例、Ⅱ度组35例、Ⅲ度组27例)以及30例正常生育且排除VC者(正 常组)的外周血生殖激素水平进行检测.结果 随着精索静脉曲张程度的加重,FSH水平逐渐升高,Ⅲ度组FSH水平([ 8.7±3.8)IU/L]与正常组([ 6.8±2.3) IU/L]比较差异有统计学意义(P<0.05),LH、PRL、T 3种生殖激素3个VC组与正常组比较,差异无统计学意义(P>0.05).结论 随着精索静脉曲张程度加 重,生殖激素水平发生异常者增多.生殖激素水平对反映精索静脉曲张所致睾丸病理生理变化有一定的参考价值.

Objective To compare the outcome of rescue intra-cytoplasmic sperm injection(ICSI) performed on patients in Group 6 h and Group 22 h after 6 h or 22 h initial in vitro fertilization(IVF).Methods Among 65 failure IVF cycles,rescue ICSI was performed on 6 h and 22 h after initial IVF retrospectively.The data of fertilization and cleavage rates,embryo quality,implantation,and pregnancy rates after rescue ICSI were analyzed and compared between two groups.Results The fertilization rate and cleavage rates in Group 6 h with 77.1% and 97.1% were higher than those in Group 22 h with 47.5% and 68.3% that the difference was significant(P0.05).Clinical pregnancies of Group 6 h was 44.0% including 18 singletons and 4 sets of twins while no clinical pregnancy was in Group 22 h.Conclusion Rescue ICSI after 6 h post-insemination has better fertilization and pregnancy rates compared with rescue ICSI after 22 h when oocytes have become aged.

Objective To discuss the methods of separation,purify and culture of neonatal mice cardiomyocytes(CM) for inducing mesenchymal stem cells(MSCs) to CM by coculture,and found the way to identify the cells.Methods CM from ventricular tissues of neonatal mice were separated with enzyme digestion,purified by differential and adherent,and treated with 5-bromodeoxyuridine(5-Brdu).The expressions of CM mRNA and protein were detected with reverse transcription-polymerase chain reaction(RT-PCR) and immunofluorescence staining,and the ultras-tructural characteristics of CM were observed with transmission electron microscope after culture for 7 days.Results CM with well morphology and spontaneous beating obtained by the use of 0.08% trypsin and 0.04% collagenase type Ⅱ,98 % cells are alive,and then CM were arranged in loose and jumping synchronously by adherent culture for 3 days.The percentage of positive cell(CM) were 99.07%,marked with anti-cardiac Troponin T(anti-cTnT) antibody by immuofluorescence staining.The expression of myocardial myosin heavy chain α/β(MHC α/β),cTnT gene in CM were detected apparently by RT-PCR with MSCs as negative control detetednegative control.The Ultra-structure of few CM can be observed,used 3%-4% Agar to pre-embed and then got the extra-thins lice.Conclusions Our studies found a way to obtain and identify neonatal mouse CM with nomal tructure and function,and provide a foundation for further inducing MSCs by cocultrue with CM,and identifying the cells after differentiation.

Objective To compare the outcomes of rescue intracytoplasmic sperm injection (ICSI) in 6 h and 22 h after conventional in vitro fertilization (IVF) to rescue unfertilized oocyte. Methods From June 2004 to November 2008, a total of 31 failure IVF cycles were included into this study. Among them, rescue ICSI was performed on 6 h after initial IVF of 15 failure IVF cycles (between June 2004 and August 2007) (6 h group), 22 h after initial IVF of 16 failure IVF cycles (between August 2007 and November 2008) (22 h group). The rates of fertilization and cleavage, embryo quality, implantation, and pregnancy rates after rescue ICSI were analyzed and compared between two groups. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Yantai Yuhuangding Hospital. There had no significant differences between two groups among average, infertility duration, and so on (P>0.05). Results The fertilization rate in 6 h group (77.8%, 70/90) was significant higher than that of 22 h group (47.5%, 58/122) (P<0.01). The cleavage rate in 6 h group (98.6%, 69/70) was also significant higher than that of 22 h group (79.3%, 46/58) (P<0.01). And the clinical pregnancy rate in 6 h group (43.8%, 7/16) was higher than that of 22 h group (0, 0/15) with significant difference (P<0.05). Conclusions It was competitive to rescue ICSI on oocytes with fertilization failure in 6 hours than 22 hours after conventional IVF. Key words: in vitro fertilization; fertilization failure; rescue intracytoplasmic sperm injection; second polar body

目的探讨结核感染T淋巴细胞斑点实验(T-SPOT.TB)在临床检测结核病中的应用价值。方法利用T-SPOT.TB方法检测结核疑似患者,进行科室分布分析,同时与抗酸杆菌涂片、结核抗体实验、结核分枝杆菌DNA检测进行对比。结果 317例结核疑似患者中,76例T-SPOT.TB阳性,阳性率24.0%。潜伏性结核筛查科室阳性率为9.1%～25%;肺结核及肺外结核检测诊断中,阳性率为20.7%～66.7%。与其它实验室检测方法对比,T-SPOT.TB方法具有明显优势。结论 T-SPOT.TB是灵敏和特异的快速检测结核分枝杆菌感染的方法,在结核疑似患者筛查与诊断中具有较大临床应用价值。