胚外体腔穿刺术应用于α地中海盆血产前诊断的探讨

目的：探讨胚外体腔穿刺术应用于α地中海贫血产前诊断的可行性。方法：对50例妊娠6－10周、要求人工流产的单胎妊娠孕妇在终止妊娠前行胚外体腔穿刺术抽吸胚外体腔液，吸宫术后取绒毛，采用聚合酶链反应（PCR）扩增胚外体腔细胞及绒毛的α地中海贫血基因，比较两种标本诊断的符合率。结果：40例胚外体腔细胞成功地扩增α地中海贫血基因，结果均与绒毛相符。结论：通过胚外体腔穿刺术取胚外体腔细胞行PCR检测可用于α地中海贫血的早期产前诊断，但应进一步提高诊断方法的敏感性。     

运用胚外体腔液产前诊断α地中海贫血的临床研究

目的探讨胚外体腔穿刺液应用于α地中海贫血产前诊断的安全性及准确性。方法对40例妊娠6～10周、要求人工流产的单胎妊娠孕妇在终止妊娠前行胚外体腔穿刺术抽吸胚外体腔液，吸官术后取绒毛，采用聚合酶链反应(PCR)检测胚外体腔细胞及绒毛的α地中海贫血基因，比较两种标本诊断的符合率。结果36例胚外体腔细胞成功地扩增α地中海贫血基因，结果均与绒毛检测结果相符。结论通过胚外体腔穿刺术取胚外体腔细胞行PCR检测可用于α地中海贫血的早期产前诊断，具有可行性。     

胚外体腔液中胎儿细胞DNA检测在产前诊断中的应用

目的探讨胚外体腔液中细胞DNA检测用于孕早期诊断性连锁遗传疾病的可行性和准确性。方法对25例孕6～11周、要求人工流产的孕妇术前行胚外体腔穿刺术,抽取胚外体腔液0．5～3ml。采用煮沸法对胚外体腔液进行DNA抽提,Picogreen染料对DNA进行定量分析。采用X,Y两对引物对DNA进行PCR检测。随后行人工流产术,取少量绒毛组织进行染色体核型分析,并与胚外体腔液中的DNA检测结果作对照。结果0．5～3ml胚外体腔液中含有750—6270个胎儿细胞。男性胎儿DNA扩增结果出现两个条带（X,Y）11例,女性胎儿出现1个条带（X）14例。与绒毛组织染色体核型分析结果完全一致。结论胚外体腔液中细胞DNA检测,用于孕早期性连锁遗传疾病的产前诊断具有快速、敏感的特点。     

α和β地中海贫血双重杂合子的基因诊断

目的 :探讨α和β地中海贫血双重杂合子的基因诊断。方法 :采用跨越缺失区断裂点的PCR方法检测α 地中海贫血 1基因。采用等位基因特异寡核苷酸探针 /反向点杂交 (ASO/RDB)技术检测 β 地中海贫血基因。 结果 :对地中海贫血筛查中发现的 3例疑为α和β地中海贫血双重杂合子进行基因诊断 ,均属于东南亚缺失型α 地中海贫血 1和β 地中海贫血双重杂合子 ,其中 1例为α 地中海贫血 1和β 2 8(A→G) ,1例为α 地中海贫血 1和βIVS Ⅱ 6 5 4 (C→T) ,1例为α 地中海贫血 1和HbE。结论 :α和 β地中海贫血双重杂合子的检出对临床准确进行地中海贫血的产前诊断有重要意义。     

应用突变引物延伸扩增技术快速产前诊断β地中海贫血基因突变

目的 探讨综合应用突变引物延伸扩增(MOEA)及巢式聚合酶链反应(PCR)技术进行β地中海贫血产前诊断的可行性.方法 先用一对外侧引物扩增1.5 Kb β珠蛋白基因片段,再用MOEA技术检测β地中海贫血点突变.结果 完成了6例β地中海贫血高危胎儿的产前基因诊断,其中双重杂合子1例,杂合子3例,正常胎儿2例;经分娩或流产后基因分析验证,结果与产前诊断完全一致.结论 MOEA技术适合于已知点突变的基因诊断和产前诊断,具有快速、简便、安全、准确等优点,便于推广应用.     

经宫颈绒毛取材用于地中海贫血产前诊断883例分析

目的:对有可能生育地中海贫血(地贫)高危胎儿的孕妇,在早孕期经宫颈绒毛活检(TC-CVS)用于地贫产前诊断,评价,TC-CVS在产前诊断中的应用价值.方法:对883例孕9～11周行地贫产前诊断的孕妇进行经宫颈绒毛活检,抽取绒毛行地贫基因分析产前诊断.随访观察孕妇的取材成功率、流产率、感染率.结果:取材成功率95.36%,自然流产率4.3%,宫内感染率1.13%.地贫基因分析共检出重型地贫儿143例,HbH病26例,α和β地贫杂合子共373例.结论:经宫颈绒毛活栓是一项安全可行的介入性产前诊断方法,用于早孕期地中海贫血产前诊断,可有效地检出重型地贫儿及各种地贫杂合子.     

孕早期胎儿地中海贫血基因型与血液学表型和产前诊断适应证的遗传研究

目的:探讨孕早期侵入性产前诊断分析地中海贫血夫妇的基因型分布与血液临床表型和产前高危因素之间的遗传关联,以更好地进行地中海贫血高风险夫妇孕早期产前诊断和遗传咨询。方法:通过对606例高风险夫妇行腹部绒毛活检术(TA-CVS)进行基因型结果分析,并依据不同的基因组合对其血液学指标血红蛋白(Hb)、平均红细胞容积(MCV)、平均红细胞血红蛋白(MCH)和血红蛋白A2(HbA2)进行比较,分析产前诊断适应证的高危风险因素与胎儿基因型临床表型结果的关系。结果:①606例绒毛标本中,地中海贫血的胎儿标本463例,其中α地中海贫血携带者共检测出基因型16种,以常见基因型--SEA/αα(东南亚缺失)为主(26.3%)。②胎儿α地中海贫血基因的Bart's和HbH病临床表型其父母血液参数呈现特点为中度且小细胞低色素贫血,且Bart's胎儿其父母血液学表型MCV、MCH、Hb水平均较HbH病低(P0.05);β双重杂合子或纯合子的血液学特点为小细胞低色素性贫血,α复合β地中海贫血和β地中海贫血杂合子血液学参数特点为轻度降低或正常,HbA2值升高或正常,且β双重杂合子或纯合子父母双方的MCV、MCH、Hb水平均显著低于相应的α复合β地中海贫血和β地中海贫血杂合子(P0.05)。③产前诊断适应证中夫妇双方高风险的高危因素中Bart's和HbH所占例数高于高龄超声异常、不良孕产史、染色体异常和重型Bart's生育史产前诊断适应证。结论:广西地区胎儿地中海贫血中以α地中海贫血东南亚缺失型发病率为主;各种胎儿基因型结果其父母血液学参数变异性较大,夫妇双方地中海贫血高风险的高危因素致地中海贫血患儿的风险系数较高,临床上应结合患者的血液学表型和产前诊断的危险因素进行判断,以避免漏诊。     

广州市黄埔区5306对夫妇孕期地中海贫血的筛查与诊断分析

目的通过对广州市黄埔区群众的地中海贫血筛查、诊断结果进行总结,了解广州市黄埔区群众地贫基因携带率,地贫类型,为本区制定地贫防治工作提供指导。方法通过对2008年1月至2012年12月在广州市黄埔区参加出生缺陷工程的5 306对夫妇孕早期抽血进行地中海贫血初筛,检查到一方或双方初筛阳性者,夫妇均进一步行地贫基因检查,对携带同型地贫基因的夫妇建议产前诊断。结果共检出地贫基因携带者1 338例,单纯性α地中海贫血801例,检出率为7.55%,单纯性β地中海贫血498例,检出率为4.69%,α复合β地中海贫血双重杂合子39例,检出率为0.37%。地贫高风险家庭48户,经产前诊断,确诊10例重型地贫胎儿,均行引产。结论黄埔区的α地中海发生率较广东其他地区稍低,但β地中海贫血发生率偏高,在预防干预时要重视;α复合β地中海贫血双重杂合子的准确检出对于做好优生优育及产前诊断非常重要。     

对遗传病高危孕妇行早期经腹绒毛取样

经腹绒毛取样可以替代经宫颈的途径进行遗传病的产前诊断。时间可由孕极早期到孕13周以后。本文研究了于孕9周前对遗传病高危孕妇行经腹绒毛取样的可行性。 在产科门诊选择200例有遗传病危险并希望得到产前诊断的孕妇。其中2例妊娠有患假肥大性肌营养不良的危险,198例有患β-地中海贫血的危险。在超声波指引下,将20-22号穿刺针以平行或轻度倾斜的方式进入丛密绒毛膜,每次取样进针不超过两次。取样后数小时孕妇即可出院,出院前须再作超声检查以确定胎儿情况,并除外绒毛膜内和绒毛膜后血肿。将绒毛DNA行聚合酶链反应得到基因扩增后,经斑点印迹分析直接检测β-地中海贫血的基因突变。用多重外显子扩增法,对一例已有一个患假肥大性肌营养不良症小孩的孕妇诊断出一个患病     

69例αβ复合型地中海贫血的血液学和基因型研究

目的:分析69例αβ复合型地中海贫血患者基因型、血液学与临床表现的关系,探索和指导这类复杂基因型组合方式的临床诊断.方法:采用标准的血液学分析技术测量红细胞参数和血红蛋白组分.反向点杂交技术诊断β地中海贫血和α地中海贫血基因点突变,缺口聚合酶链反应技术检测α-地中海贫血缺失突变.结果:45例轻型β地中海贫血合并α地中海贫血(轻型复合型)患者的临床表现与β地中海贫血携带者无异;24例重型β地中海贫血合并α地中海贫血(重型复合型)则从重度贫血到轻度贫血均有,差异较大,但多数表型为中间型地中海贫血.69例患者中,β地中海贫血的突变基因类型共检出8种,α地中海贫血的突变基因类型共检出5种.结论:轻型复合型的临床表现以轻型β地中海贫血的表现为主,α地中海贫血被掩盖;重型复合型则多表现为中间型地中海贫血.这一结果提示我们进行地中海贫血的临床检测时,尤其对β地中海贫血携带者,建议常规同时进行α地中海贫血致病基因的筛查,防止误诊、漏诊发生,进而确保地中海贫血产前诊断的准确性.     

First-trimester amniotic fluid and extraembryonic coelomic fluid acetylcholinesterase electrophoresis.

Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation.     

Prenatal paternity testing using DNA extracted from coelomic cells

OBJECTIVE: Prenatal paternity testing can be performed following invasive prenatal diagnosis with amniocentesis or CVS. Coelocentesis is a new technique that could be used as an alternative method early in the first trimester of pregnancy. The aim of this study is to investigate the potential use of the DNA extracted from coelomic cells in the prenatal paternity testing. METHODS: Coelocentesis was performed in 20 singleton pregnancies at 7-9 weeks of gestation immediately before surgical termination of pregnancy. Chorionic cells from the placenta and blood cells from the parents were processed by the standard salt extraction method. Two loci, TPO and Apo B, were used for paternity testing in the DNA of coelomic cells, chorionic cells and blood cells. RESULTS: There was concordance in the results obtained from the coelomic cells and chorionic villi. In two cases only the polymorphisms used were not conclusively informative for paternity exclusion. CONCLUSIONS: Coelomic cells are potentially useful for early paternity testing.     

Aneuploidy screening in coelomic samples using fluorescence in situ hybridisation (FISH)

OBJECTIVES: Coelocentesis is the earliest invasive prenatal diagnostic procedure that has recently been used in ongoing pregnancies to identify single gene defects. Aneuploidy screening has not yet been performed in ongoing pregnancies following coelocentesis, but experimental studies have demonstrated the ability of determining the copy number of chromosomes 13, 18, 21, X and Y in uncultured coelomic samples, by FISH or PCR. The aim of this study was to extend previous studies and investigate the feasibility of analysing uncultured coelomic fluid samples for 11 chromosomes using fluorescence in situ hybridisation (FISH). METHODS: Coelocentesis was performed in 12 singleton pregnancies at 6 to 9 weeks of gestation immediately before surgical termination of pregnancy. Fluorescence probes for chromosomes 3,7,9,13,16,17,18,21,22, X and Y were applied on uncultured coelomic-fluid samples and placental tissue. In cases where coelomic cells were not of a sufficient amount, chromosomes X and Y were analysed in a second layer of hybridisation. RESULTS: Successful analysis by FISH was possible in all cases and the results from the coelomic fluid were concordant with those from the analysis of placental tissue and obtained within a few hours of receiving the samples. Problems associated with limited cell numbers were overcome by the application of a second layer of FISH. This sequential approach has also enabled accurate identification of maternal-cell contamination in male samples. CONCLUSION: Analysis of 11 chromosomes using FISH in coelomic fluid samples is feasible and it has the potential to be applied for rapid aneuploidy screening, should coelocentesis be used clinically as an early, invasive prenatal diagnostic tool.     

Biochemical composition of amniotic fluid and extraembryonic coelomic fluid in the first trimester of pregnancy.

OBJECTIVE: To determine the biochemical composition of amniotic fluid and extraembryonic coelomic fluid between 8 and 12 weeks gestation. DESIGN: Prospective observational study. SUBJECTS: 40 women with a normal pregnancy between 7 and 12 weeks gestation having termination of pregnancy. INTERVENTIONS: Before termination the women had a transvaginal ultrasound guided amniocentesis. Pure samples of amniotic fluid and extraembryonic coelomic fluid were obtained from each woman and standard biochemical variables were measured in each fluid sample immediately after collection. RESULTS: Levels of sodium, potassium and bicarbonate were significantly higher in amniotic fluid whilst chloride, urea, bilirubin, protein, albumin, glucose, creatinine, calcium and phosphate were present in higher concentrations in extraembryonic coelomic fluid. All differences in concentration were significant (P less than 0.05; unpaired t-test). No relation was demonstrated between electrolyte concentrations in amniotic fluid or coelomic fluid and stage of gestation. CONCLUSIONS: Amniotic fluid and extraembryonic coelomic fluid have a widely differing biochemical composition. The biological significance of these differences remains unexplained.     

Erythropoietin levels in amniotic fluid and extraembryonic coelomic fluid in the first trimester of pregnancy.

OBJECTIVE: The aim was to measure erythropoietin levels in amniotic fluid and extraembryonic coelomic fluid from 7-12 weeks' gestation. SUBJECTS: Twenty healthy women with ultrasonographically normal first trimester pregnancies prior to surgical termination. METHODS: Paired samples of amniotic fluid and extraembryonic coelomic fluid were collected by transvaginal ultrasound guided needling. Erythropoietin was measured in both pregnancy fluids using a radioimmunoassay. RESULTS: There was a highly significant difference between erythropoietin levels in extraembryonic coelomic fluid (median level 15.45 mU/ml; range 6.8-32.1 mU/ml) and those in amniotic fluid (median 5.0 mU/ml; range < 5.0-5.8 mU/ml) (P < 0.0001; Mann-Whitney U-test). The levels of erythropoietin in maternal serum (median 15.4 mU/ml; range 5.6-29.4 mU/ml) were similar to those in the extra-embryonic coelom (P = 0.81; Mann-Whitney U-test). No relation was demonstrated between erythropoietin levels in amniotic fluid or coelomic fluid and stage of gestation. CONCLUSION: High levels of erythropoietin in coelomic fluid suggests that the hormone is involved in the process of human extraembryonic erythropoiesis. The exact regulatory role remains unknown.     

Early amniocentesis: alphafetoprotein levels in amniotic fluid, extraembryonic coelomic fluid and maternal serum between 8 and 13 weeks.

OBJECTIVE--The aim was to establish a normal range of alphafetoprotein (AFP) concentrations in amniotic fluid from 8 to 12 weeks gestation, and to determine any difference between AFP levels in amniotic fluid and extraembryonic coelomic fluid. DESIGN AND SUBJECTS--150 women had a transvaginal ultrasound guided amniocentesis before termination of an apparently normal first trimester pregnancy. Separately identified samples of amniotic fluid and extraembryonic coelomic fluid were obtained and assayed by radioimmunoassay for AFP. RESULTS--In amniotic fluid, very high levels of AFP were present at 8 weeks, levels falling rapidly up to 10 weeks after which there was a slight rise. Thus over the period 8 to 10 weeks, there was a significant inverse correlation between amniotic fluid AFP and gestational age (r = 0.67; P less than 0.001). In extraembryonic coelomic fluid, by contrast there was no trend in AFP relative to gestational age. CONCLUSIONS--The rapidly changing levels of AFP from 8 to 10 weeks as well as the small volume of the amniotic cavity makes the use of amniocentesis impracticable before 11 weeks gestation. The lack of any relation between AFP levels in amniotic fluid and extraembryonic coelomic fluid emphasises the importance of identifying the site of amniocentesis in the first trimester.     

Human chorionic gonadotrophin and alpha-fetoprotein levels in matched samples of amniotic fluid, extraembryonic coelomic fluid, and maternal serum in the first trimester of pregnancy.

Separately identified samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 32 women between 7 and 12 weeks of pregnancy were analysed for human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP). There was a highly significant difference between the hCG levels in amniotic fluid (median level 6.3 U/ml; range 1.6-310.0 U/ml) and those in extraembryonic coelomic fluid (median level 400.0 U/ml; range 135.0-2250.0 U/ml) (p less than 0.001; Mann-Whitney U-test). The levels of AFP were very similar in amniotic fluid (median 26.0 kU/ml; range 10.0-116.5 kU/ml) and extraembryonic coelomic fluid (median level 24.1 kU/ml; range 12.4-94.4 kU/ml).     

The coelomic cavity — A reservoir for metals

OBJECTIVE: The concentrations of metals in fluids surrounding the first trimester fetus were measured.STUDY DESIGN: Atomic absorption spectrometry was used to measure concentrations of metals in matched samples of amniotic and extraembryonic coelomic fluids in 17 women between 9 and 12 weeks of pregnancy.RESULTS: Concentrations of calcium, magnesium, iron, copper, and manganese (but not zinc, cadmium, or lead) were significantly higher in coelomic than in amniotic fluid. There was no significant difference between levels of iron, manganese, and lead to controls and amniotic fluid or between concentrations of manganese, cadmium, and lead in controls and coelomic fluid. There was no relationship between the concentrations of each metal in amniotic and coelomic fluid.CONCLUSION: The extraembryonic coelom is an important site of concentration of metals in early pregnancy. This might represent a store of metals essential for normal embryonic and fetal development or constitute a defense mechanism against environmental conditions adverse to the fetus.