Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.     

Dydrogesterone in the reduction of recurrent spontaneous abortion

One hundred and eighty Women with a history of recurrent, unexplained spontaneous abortion (mean 3.5 abortions) were randomised to receive oral dydrogesterone (10 mg b.i.d.), intramuscular human chorionic gonadotrophin (hCG; 5000 IU every 4 days) or no additional treatment (controls). Treatment was started as soon as possible after confirmation of pregnancy and continued until the 12th gestational week. All women received standard supportive care. Abortions were significantly (p ≤ 0.05) less common in the dydrogesterone group (13.4%) than in the control group (29%); there were no statistically significant differences between the hCG group and the control group. There were no differences between the groups with respect to pregnancy complications or congenital abnormalities. In conclusion, hormonal support with dydrogesterone can increase the chances of a successful pregnancy in women with a history of recurrent spontaneous abortion.     

Placental implications for pregnancy complications in the chimpanzee (Pan troglodytes)

Placentas from 28 term chimpanzee pregnancies, including two sets of dichorionic-diamniotic twin-pregnancies, were examined and compared histopathologically with those obtained from 171 small-for-dates (SFD), 306 premature, and 77 pregnancy toxemic human infants. Term infant and placental weights for the chimpanzee were generally smaller than for any of the human categories studied. Macroscopically, the chimpanzee placentas showed a high frequency of extrachorialis, infarctions, intervillous thrombi, and marginal hemorrhages, pathologies frequently associated with pregnancy toxemia and abruptio placenta in the human being. Yet, ultrastructurally, the chimpanzee chorionic villi evidenced well-developed organella, syncytial microvilli, and chorionic capillaries, although villitis and inflammation of the membranes and cord were not infrequently seen. These findings suggest that chimpanzees may suffer the same obstetric complications seen in human pregnancies.     

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures

Background aimsFirst-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.MethodsWe set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.ResultsGrowth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.ConclusionsOur findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.     

Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy

Summary Morphological changes in fixed stromal cells and Hofbauer cells were studied throughout pregnancy in different types of placental chorionic villi by scanning electron microscopy. In the mesenchymal villus the fixed stromal cells were characterized by thin cytoplasmic processes. Hofbauer cells exhibited blebs on their surface. Large sail-like processes with a crescent profile which surrounded well developed stromal channels and a small cell body typified the small reticulum cells of the immature intermediate villus. The Hofbauer cells here displayed blebs, microplicae and large lamellipodia. Short cytoplasmic expansions and a large cell body characterized the fibroblasts present inside the stem villus. Hofbauer cells were rare, having blebs or a few short lamellipodia. The mature intermediate villus contained small and large reticulum cells. The latter had a much larger cell body than the small ones and displayed a few short cytoplasmic processes partly delimiting narrow incomplete stromal channels. Occasional Hofbauer cells with small microplicae and/or blebs were present. The small reticulum cells and fibroblasts present in the terminal villus showed similar morphological features as above. However, the former exhibited less developed cytoplasmic extensions and therefore no stromal channels were observed. In the terminal villus, the morphology of the rare Hofbauer cells was similar to that found in the mature intermediate villus.     

孕激素及人绒毛促性腺激素与药物流产后异常子宫出血的关系

目的:分析孕激素和人绒毛膜促性腺激素(h CG)与药物流产后异常子宫出血的关系。方法:选择2017年1月至2017年12月我院妇产科收治的药物终止妊娠的妇女150例,患者口服米非司酮配伍米索前列醇药物终止早期妊娠。将药物流产后子宫出血时间≤14 d作为对照组(n=75),14d作为异常组(n=75)。比较两组患者在药物流产后10 d、14 d、18 d、22 d血清中孕激素和h CG含量,分析两组患者孕激素和h CG含量相关性。结果:两组患者在年龄、月经周期、孕次、受孕天数、体重等方面比较无统计学差异(P0.05)。异常组在药物流产后10 d、14 d、18 d、22 d血清孕激素和h CG含量均高于对照组(P0.05)。两组患者在药物流产后10 d、14 d、18 d、22 d孕激素含量呈先降低再升高的趋势(P0.05)。对照组患者在药物流产后10 d、14 d、18 d、22 d血清hCG含量逐渐降低(P0.05);异常组在药物流产后10 d、14 d血清h CG含量比较无统计学差异(P0.05),在药物流产后18 d、22 d血清hCG含量低于药物流产后10 d、14 d,且药物流产后22 d低于药物流产后18 d(P0.05)。对全部样本的全部时点数据合并进行Pearson相关检验分析,孕激素和h CG含量呈正相关关系(P0.05)。结论:药物流产后异常子宫出血妇女血清的孕激素、hCG含量较高,两者呈正相关关系。药物流产后10 d、14 d监测血清HCG值无明显下降提示有异常子宫出血的可能,联合监测孕激素、hCG含量有利于药物流产后异常子宫出血的预测和治疗。     

子宫动脉血流参数联合血清β-HCG、P、E2预测复发性流产再次妊娠孕妇流产的价值研究

目的：探讨子宫动脉血流参数[搏动指数(PI)、阻力指数(RI)、收缩期/舒张期血流速度(S/D)]联合血清β-人绒毛膜促性腺激素(β-HCG)、孕酮(P)、雌二醇(E₂)预测复发性流产(RSA)再次妊娠孕妇流产的价值。方法：选取2021年1月～2022年10月安徽省妇幼保健院收治的RSA再次妊娠孕妇145名(RSA组),另选取同期我院145名健康孕妇(对照组),根据妊娠结局将RSA再次妊娠孕妇分为流产组(65例)和活产组(80例)。检测血清β-HCG、P、E₂水平,并采用经阴道超声检测子宫动脉血流参数。多因素Logistic回归分析影响RSA再次妊娠孕妇流产的因素,受试者工作特征(ROC)曲线分析子宫动脉血流参数联合血清β-HCG、P、E₂预测RSA再次妊娠孕妇流产的价值。结果：RSA组PI、RI、S/D高于对照组,血清β-HCG、P、E₂水平低于对照组(P<0.05)。流产组PI、RI、S/D高于活产组,血清β-HCG、P、E₂水平低于活产组(P<0.05)。多因...     

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.     

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery,viability and T-cell function

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality.     

用绒毛组织微核评价母亲因素对人胚的致突变性

对507对欲作人工流产的夫妇进行断面调查及绒毛组织微核的测定,以探讨父母吸烟、饮酒及采取避孕措施等因素对子代的致突变性。结果显示:微核的发生率与母亲年龄、孕次、孕龄、既往流产史等自身的因素无关;各种避孕措施及少量饮酒均不具有诱发绒毛微核细胞率增加的作用。然而,男方吸烟、女方被动吸烟其微核细胞率为0.7645±0.0561‰高于饮酒组(0.5667±0.2004‰)和非吸烟对照组(05522±0.0616‰),与对照组有显著性统计学意义(P<0.05)。男方配偶吸烟兼饮酒者其微核发生率最高(0.7944±0.0754‰),说明吸烟与饮酒对诱发绒毛微核的产生有协同作用的趋势。本文并对男方吸烟和/或女方被动吸烟可诱发胚胎绒毛组织细胞DNA和/或纺锤体装置的损伤,进行了探讨,提出应引起人们的高度重视,并建议利用人类绒毛组织微核测定法作为监测环境致突变因子对人类下一代影响的方法。     

深部热水硫酸盐还原菌微滴数字PCR检测技术的建立与应用

[背景]地下深部存在一个生物圈,深部沉积岩、玄武岩、花岗岩和变质岩等岩性环境的微生物群落已被调查,而地下深部碳酸盐岩岩溶-裂隙热储层微生物群落特征仍然不清.硫酸盐还原菌(sulfate-reducing bacteria,SRB)是地下深部频繁检出的微生物.[目的]建立快速准确定量深部热水硫酸盐还原菌的微滴数字PCR ...     

家鸽小肠绒毛微血管铸型的扫描电镜观察

用扫描电镜观察了ＡＢＳ丁酮溶液灌注的家鸽小肠绒告发同血管构筑情况。家鸽小肠绒毛血管丛由输入沁动脉、毛细血管网和输出小静脉组成,小肠绒毛血管丰富,并相到吻合成单层密集网;办入小动脉既可从肠腺周围血管丛发出,也可直接由粘膜下去一发出,绒毛下部血管表现为微直血管形态,可能部分具有门静脉性质。     

SD-PMA-ddPCR检测食品中单核细胞增生李斯特氏菌

【目的】检测食品中单核细胞增生李斯特氏菌活菌。【方法】利用脱氧胆酸钠(SD)对受损细胞预处理,然后使叠氮溴化丙锭(PMA)进入受损细胞与DNA发生共价交联,提取细菌基因组DNA进行微滴式数字PCR(dd PCR)检测。【结果】0.1%SD和5.0 mg/L PMA协同作用,可以有效抑制108 CFU/m L的单核细胞增生李斯特氏菌死菌DNA的PCR扩增。经过SD和PMA对样品预处理,dd PCR可以在死菌存在条件下,定量检测鸡肉中单核细胞增生李斯特氏菌活菌,消除了\"假阳性\"结果的出现。活菌灵敏度检测结果显示:SD-PMA-dd PCR的灵敏度为2.0 copies/20μL。SD-PMA-dd PCR方法精密度和稳定性良好。【结论】SD-PMA-dd PCR在检测食源性致病菌方面有巨大的发展空间。     

Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA

Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland–Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.