Enhancement of amniotic fluid cell growth for genetic amniocentesis

The last two decades have witnessed a logarithmic growth in demand for prenatal diagnosis of human disease through amniocentesis. Consequently, culture turn-around-time has become the major concern of all those who are primary care providers for patients seeking help. We report a rapid method for obtaining cytogenetic results from amniocytes as early as 5 days. Early tapping and reducing the turn-around-time during cytogenetic analysis may provide an alternative to chorionic villi sampling.     

提高羊水细胞培养成功率的方法

目的提高羊水培养的成功率。方法正常羊水保留换液标本,以备复查;异常羊水采用细胞分离技术提取羊水中的有效细胞成分;及时发现污染羊水并进行抗菌处理。结果提高了羊水培养的成功率（99.4%）并缩短异常羊水的培养时间,解决了部分污染羊水的污染问题（2/3）。结论保留换液标本可有效解决因收集不当而导致无法诊断的问题;细胞分离技术可提高异常羊水细胞培养的成功率并缩短培养时间,污染的羊水也可抗菌后培养成功。     

Prenatal cytogenetic diagnosis: First 1,000 successful cases

From February 1969 to August 1976, we studied 1,048 amniotic fluids. Of these, 958 (91.4%) were primarily for prenatal cytogenetic diagnosis. Cytogenetic studies were attempted in 1,021 cases; the diagnosis was successful in 1,000 of these. The failure rate of obtaining a diagnosis from the amniotic fluid cell culture of the first amniocentesis was 5% (50 cases); 29 cases had a repeat tap and successful diagnosis was achieved in all. In 21 cases, a repeat tap was refused. Thus, the overall failure rate of obtaining a final cytogenetic diagnosis was 2.06% (21/1,021). There were 32 fetal losses after amniocentesis including 16 spontaneous second trimester abortions, 7 fetal deaths in utero and 9 stillbirths. In two additional cases, fetal death had occurred before amniocentesis. This number of fetal losses does not exceed the number that would be expected in the same maternal age group without amniocentesis. In our series, the frequencies of trisomy in maternal age groups 40 years and over, 37–39 years, 35–36 years, and under 35 years were 4.5, 3.14, 0 and 0% respectively. These frequencies are comparable to those reported from other prospective prenatal studies and higher than those of retrospective live born studies. Various problems and pitfalls in prenatal cytogenetic diagnosis are discussed.     

Mosaicism in amniotic fluid cell cultures: Classification and significance

The last decade has witnessed increasing application of human cytogenetic technology to prenatal chromosome analysis. However, unlike the rather uniform peripheral blood T-lymphocyte system which has provided most of our experience in human cytogenetics, long-term amniotic-fluid cell cultures display extreme cellular heterogeneity and disproportionate growth of certain cell types as a consequence of clonal amplification. When they enter cell culture, many of these cells are approching the terminal stages of their respective life spans and may have accumulated chromosomal aberrations. Concern about the possibility of true fetal mosaicism seems warranted chiefly in situations were multiple colonies display potentially viable aberrations. Clonal analysis, preferable of multiple clonal types, and attention to details of clonal morphology are likely to minimize diagnostic errors and undue apprehension resulting from mosaicism in amniotic-fluid cell cultures.     

Amniotic fluid cell culture for prenatal diagnosis of some metabolic diseases

Primary cultures of amniotic fluid cells were obtained. Optimal conditions of culture were determined and the mean incubation period was 17.9 days. Normal values for the activity of eight glycosidases, deficiency of which causes the development of inborn errors of metabolism, were obtained. On the basis of the results it is concluded that glycolipidoses (Tay-Sachs', Fabry's, and Gaucher's diseases, etc.) can be diagnosed prenatally. Data on the prenatal diagnosis of Tay-Sachs' disease are presented.Department of Medical Genetics, All-Union Research Institute of Obstetrics and Gynecology, Ministry of Health of the USSR, Moscow. Laboratory of Biochemistry and Pathochemistry of Carbohydrate Metabolism, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. S. Persianinov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 344–346, March, 1980.     

孕中期羊水中细胞研究进展

孕中期羊水中细胞培养和核型鉴定广泛应用于产前诊断领域,可以发现一系列胎儿发育异常。近来,许多研究表明羊水中含有胎儿的干细胞,这些细胞能否应用于干细胞和组织工程学领域成为研究热点。综述了羊水中细胞的组成、来源、生物学特性、干细胞特性及组织工程学应用等方面的内容,并对研究前景进行展望。     

Metaphase chromosomes from primary culture of human amniotic fluid cells

Summary A new growth medium is specially designed for reliable and reproducible primary culture of human amniotic fluid cells. In comparison with many conventional media at 15 to 20% serum, this medium at 4% serum provides actively growing primary cultures for cytogenetic studies in less time with a larger number of metaphase cells present.     

Lactoferrin in human amniotic fluid

The concentration of human lactoferrin (LF) was measured byradioimmunoassay or non-competitive avidin-biotin assay in amnioticfluid, cord blood and in the decidua, trophoblast, fetal membranesand umbilical cord. Amniotic fluid was obtained by amniocentesis,and cord blood and tissue samples were taken after deliveryor elective Caesarean section. No detectable concentration ofLF was found in amniotic fluid before week 20 of pregnancy.A significant increase in the LF concentration was observedaround week 30 and it remained high until term. In cord blood,an undetectable or low concentration of LF was measured. Intissue specimens the amount of LF was highest in the decidua(9–95µ/g a moderate concentration was assayed inthe amniotic, (2–37µg/g) and chorion (2–26µg/g) membrane and in the trophoblast (5–35 µg/g).In the umbilical cord, the concentration was > 1µg/g.These results suggest a decidual origin of LF. The role of LFduring pregnancy is discussed     

Familial amniotic bands

Amniotic bands can cause a wide variety of deformities and mutilations. They are generally considered sporadic. Two families are presented with apparent familial amniotic band anomalies. Additional cases in the literature are reviewed. These aggregations may be coincidental, and recurrence risk is apparently low. However, amniotic band malformations may be an indication for caution in the use of amniocentesis in future pregnancies.     

A high incidence of maternal cell contamination of amniotic fluid cell cultures

Maternal cell contamination (MCC) of amniotic fluid cell cultures was detected as an admixture of male and female cells in 15 out of 3,000 cases. Allowing for undetected cases (in which the fetal karyotype is female), the true incidence of MCC may be as high as 1%. Cultures affected by MCC were, in general, established from fluid samples without obvious blood contamination. No common obstetrical or amniocentesis procedural factors could be recognized among those cases affected by MCC. The proportion of maternal cells was highly variable and in some instances maternal cells were present in multiple cultures. In this series, MCC has not led to any cytogenetic misdiagnoses. MCC may pose its greatest danger for prenatal biochemical diagnoses where the properties of an entire cell population are determined.     

羊水来源胎儿间充质干细胞定向诱导为脂肪细胞的研究

目的:研究体外诱导孕中期羊水来源胎儿间质干细胞(M SCs)分化为脂肪细胞。方法:机械手段挑取孕中期羊水细胞培养体系中的胎儿M SCs集落,培养扩增后,通过流式细胞仪检测表面抗原表达进行鉴定。取第3代胎儿M SCs,IBM X、胰岛素、氢化考的松等诱导其向脂肪细胞分化。倒置相差显微镜观察诱导后细胞形态的变化。诱导20 d后,油红O染色检测胞内中性脂肪并计算阳性细胞率,W estern Blot蛋白印记法检测PPA Rγ的表达。结果:机械手段分离出的胎儿M SCs为CD 44阳性,CD 34、CD 45、H LA-D R阴性,符合M SCs特点。诱导72 h后,细胞内有脂滴出现,随着时间延长,脂滴逐渐增加并融合为脂泡,细胞由梭形转变为圆形或多角性。20 d后85%以上胎儿M SCs变为油红O染色阳性,W estern Blot显示诱导20 d后,诱导组细胞PPA Rγ的表达量明显高于未诱导组。结论:经上述方法诱导20 d后,羊水来源胎儿M SCs可以分化为脂肪细胞。     

Open neural tube defects: Immunocytochemical demonstration of neuroepithelial cells in amniotic fluid

Cytologic evaluation of second trimester amniotic fluid (AF) is a rapid, inexpensive adjunct to prenatal diagnosis of open neural tube defects (ONTDs). Our goal was to determine whether the neural-appearing cells and/or large foamy macrophages in the AF of anencephalics are indeed of neural and/or glial origin. In two second trimester patients with elevated serum alpha-fetoprotein (AFP) and polyhydramnios, fetal sonogram studies showed anencephaly; amniocentesis was performed for AF-AFP, cytogenetic, and cytologic studies. AF sediment smears were initially Papanicolaou-stained; next, the same smears were immunoperoxidase (IP)-stained for glial fibrillary acidic protein (GFAP). If GFAP negative, slides were restained for synaptophysin (SYN) and neuron-specific enolase (NSE). Both AFs contained small neural-appearing cells (5–10 μm) singly and in clusters, with dense, round, homogeneous nuclei, an occasional nucleolus, and scant cytoplasmic rim. These were GFAP negative and SYN and NSE positive; the large vacuolated, lipid-laden macrophages (20–40 μm) were negative for all three IP stains. In conclusion, positive IP staining for SYN and NSE supports the morphologic impression that small dark cells in AF are of neural origin, while negative IP staining of large foamy macrophages suggests nonneural, nonglial origin. Diagn. Cytopathol. 16:143–144, 1997. © 1997 Wiley-Liss, Inc.     

羊水干细胞定向及稳定分化为神经元样细胞体系的建立

文题释义： 羊水干细胞：羊水是由多种细胞组成的异质细胞群,其中包括胎儿脱落的表皮细胞、呼吸道上皮细胞、消化道和泌尿道等体腔管道的脱落细胞、羊膜脱落细胞等。羊水中包含有向内、中、外3个胚层分化潜能的干细胞,约占1%,近年来研究发现羊水干细胞在体外通过特殊的诱导微环境能够分化成多种不同类型的细胞,使羊水来源干细胞成为干细胞研究的一个新热点。 悬滴培养：与传统的二维培养模式不同,悬滴培养提供的三维环境有利于促进各种干细胞的增殖与分化,可以使细胞聚集,相互紧密接触,从而促进细胞之间的相互作用,进而提高或增强干细胞的生物学功能。羊水干细胞悬滴培养可形成拟胚体结构,具有较强的分化潜能,有助于提高定向诱导分化效率。 背景：利用干细胞分化代替受损神经元的设想,已经逐渐受到科研工作者的关注。胚胎干细胞和诱导性多能干细胞虽然具有良好的神经元分化潜能,但由于接种活体动物具有致瘤性,限制了其进一步的深入研究。 目的：建立稳定的羊水干细胞分选、培养、成神经诱导分化体系,探讨其作为神经元再生种子细胞的可行性。 方法：经B超引导下,穿刺获取孕19-22周期间的羊水样本10 mL,磁珠分选其中c-Kit阳性的羊水干细胞。免疫荧光染色鉴定羊水干细胞标记物Oct-4、Sox2;RT-PCR检测羊水干细胞多次传代后干性标记物c-Kit、Oct-4、Sox2、Nestin的表达;羊水干细胞悬滴培养4 d观察拟胚体的形成情况;采用“两阶段”分步诱导羊水干细胞向神经元方向分化,免疫荧光染色观察Neuro D、Tuj1的表达。 结果与结论：①羊水中可分选出大约1%的c-Kit阳性羊水干细胞;②(75.0±4.6)%的羊水干细胞表达Oct-4,(86.0±2.8)%的羊水干细胞表达Sox2;③RT-PCR方法检测干性标记物c-Kit、Oct-4、Sox2、Nestin的表达量并不随细胞传代数的增加而改变;④经悬滴培养可形成拟胚体并表达Oct-4;⑤免疫荧光证实未经诱导的羊水干细胞表达神经元标记物Tuj1,但缺乏典型的神经元形态特征;RT-PCR证实不同羊水干细胞标本都能检测到Tuj1的表达,同一样本多次传代也能稳定检测到Tuj1的表达;⑥羊水干细胞经碱性成纤维细胞生长因子、脑源性神经营养因子、神经营养因子3的两阶段分步诱导后表达神经干细胞标记物Neuro D和神经元标记物Tuj1,具有神经元样细胞的形态特征。 ORCID: 0000-0001-6488-2055(宗凌) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Chorionic plate vessels as an origin of amniotic fluid neutrophils

The present study was conducted to investigate the potential anatomical source of amniotic fluid neutrophils. Microdissection of neutrophils from the chorioamnion of the fetal membranes and the amnion of the chorionic plates of 10 preterm placentas with acute chorioamnionitis was performed and the genotypes of the neutrophils were compared with those of the mother and fetus using polymerase chain reaction of nine autosomal STR loci. In separate analyses, we reviewed eight cases of fetal autopsies with increased amniotic fluid neutrophils for the presence of neutrophils in the alveoli, and also analyzed the relationship between the amniotic fluid white blood cell (WBC) count and the histological pattern of placental inflammation. The genotypes of all of the neutrophils found in the chorioamnion of the fetal membrane matched those of the mother (n = 10). The genotypes of neutrophils found in the chorionic plate were of mixed maternal and fetal origin (n = 4). In the autopsy series of the fetuses with amniotic fluid WBC (n = 8), only five cases showed neutrophils in the alveolar space, while all the placentas had chorioamnionitis. There was no significant difference in amniotic fluid WBC count between the cases with or without acute membranitis, while among the cases with placental inflammation, those with inflammation of the chorionic plate had a significantly higher amniotic fluid WBC count than both the membranitis-only cases (P < 0.001) and the membranitis and funisitis cases (P < 0.05). These results imply that fetal vasculature at the chorionic plate is the main source of amniotic fluid neutrophils, especially in the cases without funisitis.     

羊水偏少与围产儿结局的临床研究

目的探讨羊水偏少孕妇发生胎儿不良结局的危险.方法 2002年8月～2002年12月在四川省人民医院产科住院分娩的产妇202例,羊水指数(AFI)测量8.1～20cm为羊水量正常,5.1～8.0cm为羊水偏少.围产儿不良结局包括胎儿宫内窘迫、5分钟Apgar评分≤7、胎儿生长受限(FGR)、死胎、胎儿畸形发生率.结果围产儿不良结局的发生率羊水偏少组较正常组发生率增高,FGR发生率较正常组增加两倍.结论产前检查羊水偏少患者发生围产儿不良结局及FGR的风险增高.     

Uncultured cells in amniotic fluid from normal and abnormal foetuses

Direct smears of uncultured amniotic fluid cells were analysed in 41 pregnancies between 17 and 22 weeks of gestation. These included 27 normal pregnancies where a healthy child was born at term, six anencephalic and five spina bifida pregnancies, and three other abnormal pregnancies. In normal amniotic fluid, epithelioid cells predominated but small numbers of histiocyte-like cells, typical macrophages and elongated cells were also present. The proportion of macrophages and elongated cells was significantly increased in all anencephalic cases. Two of the spina bifida cases also showed abnormal cellular proportions. Haemosiderin and lipid material was demonstrated in some of the cellular proportions. Haemosiderin and lipid material was demonstrated in some of the macrophages. Tissue smears and histological preparations suggest that these cells may originate from both the amnion and the fibrovascular membrane covering the neural lesion. This direct method of observation of amniotic fluid cells, not involving culture techniques, could provide a rapid means of prenatal diagnosis of anencephaly, and may help in the diagnosis of spina bifida.     

Chronic chorioamnionitis displays distinct alterations of the amniotic fluid proteome

Acute chorioamnionitis of infectious origin and chronic chorioamnionitis of immunological origin are two major placental lesions of spontaneous preterm birth with elevated amniotic fluid interleukin-6 and CXCL10 concentrations, respectively. The changes in the amniotic fluid proteome associated with intra-amniotic infection and acute chorioamnionitis are well defined, yet alterations unique to chronic chorioamnionitis remain to be elucidated. This study was conducted to determine those amniotic fluid proteins changing specifically in the presence of chronic chorioamnionitis. Amniotic fluid obtained from acute chorioamnionitis, chronic chorioamnionitis and gestational age-matched controls were analysed by two-dimensional (2D) difference in gel electrophoresis and MALDI-TOF analyses. The type of histological inflammation was used to define each condition in preterm labour cases (n = 125) and term not in labour cases (n = 22), and the amniotic fluid concentrations of interleukin-6, CXCL8, CXCL10 and prostaglandin F(2α) were also measured by specific immunoassays. Among preterm labour cases, 31 differentially expressed proteins were identified in chronic chorioamnionitis cases as compared to both acute chorioamnionitis and control cases. Importantly, glycodelin-A, which maintains maternal tolerance against an allogeneic fetus, was decreased in chronic chorioamnionitis, while haptoglobin was increased. We report the amniotic fluid proteome of chronic chorioamnionitis for the first time, and the findings herein strongly suggest that there is a pathophysiological association between the changes of immunomodulatory proteins in the amniotic fluid and chronic chorioamnionitis, a histological manifestation of maternal anti-fetal allograft rejection.     

兰州地区2411例孕妇羊水细胞染色体核型产前诊断分析

目的探讨羊水细胞培养染色体核型分析技术在细胞遗传学产前诊断中的应用及意义。方法2411例妊娠17～26周的孕妇在超声引导下行羊膜腔穿刺术,进行细胞培养及染色体核型分析。结果2411例标本一次性培养成功2391例,培养成功率为99．2％。共检出异常核型93例,异常率为3．89％,其中21-三体40例,18-三体11例,其他异常核型42例。结论羊水细胞学检查作为一项产前诊断技术对于指导优生优育,降低缺陷儿的出生具有重要意义。