CNVplex联合STR技术在复发性流产遗传学查因中的应用

目的 运用快速检测绒毛染色体拷贝数变化的方法探讨复发性流产查因途径.方法 对60例反复自然流产病例的绒毛样本(孕13周前)分别进行多重DNA拷贝数(CNVplex)和荧光原位杂交(FISH)技术检测,所有样本同时行绒毛培养核型分析验证.结果 48例培养成功的复发性流产病例,染色体异常率为60.42％.其中48例CNVplex检测结果与核型分析相一致:包括20例染色体正常者、23例常染色体三体,3例三倍体和2例45,XO(Turner综合征);而FISH检测结果中与核型分析结果相符的只有38例.对于非嵌合体和非结构异常样本,两种方法与细胞遗传学核型分析结果符合率分别为100％ (CNVplex)和79.17％ (FISH).结论 应用CNVplex联合短串联重复序列(STR)技术可检测绒毛染色体拷贝数及检测5 Mbp以上的重复和缺失异常,利于分析流产病因.     

Low fetal loss rates after ultrasound-proved viability in early pregnancy

Once pregnancy is recognized clinically, it is accepted that 12% to 15% undergo spontaneous abortion. However, the actual time of fetal demise has not yet been determined. To address this question, the outcomes of pregnancies identified before 21 days of conception by serum beta-human chorionic gonadotropin assays were studied. All subjects underwent ultrasound examinations at eight and 12 weeks' gestation. Among 220 women who had a viable pregnancy at eight weeks, only seven (3.2%) experienced a fetal loss thereafter. The results of this study suggest that most clinically recognized spontaneous abortions manifested after eight weeks actually represent pregnancies in which fetal demise occurred before eight weeks. These findings have important implications with respect to the safety of chorionic villi sampling and to the identification of exogenous agents that cause fetal wastage.     

组织蛋白酶B和L在早期妊娠绒毛及蜕膜中的表达

目的 观察组织蛋白酶B(cathepsinB,CB)和L(cathepsinL,CL)在人早期妊娠绒毛及蜕膜中的表达,探讨它们在胚胎着床及发育过程中的作用。方法 研究组包括:正常早期妊娠行人工流产者30例、早期自然流产者25例、正常分泌期子宫内膜10例和葡萄胎15例。应用免疫组织化学法检测CB和CL的表达。结果 CB、CL主要阳性表达于正常早期妊娠滋养层细胞和蜕膜细胞。CB在正常分泌期内膜、正常早期妊娠蜕膜、自然流产蜕膜中的阳性率表达分别为10%(1/10)、83.3%(25/30)、32.0%(8/25);CL则分别为0、63.3%(19/30)、32.0%(8/25);CB和CL在3组间的表达有显著性差异(P<0.05)。CB在早期妊娠绒毛、自然流产绒毛、葡萄胎组织中表达强阳性率分别为10.0%(3/30)、0、66.7%(10/15);CL则分别为26.7%(8/30)、4.0%(1/25)、80.0%(12/15)。CB和CL在3组间的表达有显著性差异(P<0.05)。结论 CB和CL在正常和异常妊娠蜕膜和绒毛中的表达差异提示它们胚胎着床过程中可能起着很重要的作用。     

用荧光原位杂交技术快速诊断孕早期胚胎染色体数目异常

目的探讨荧光原位杂交(FISH)技术在快速产前诊断孕早期胚胎染色体数目异常中的价值.方法采用13,18,21,X和Y染色体特异性DNA探针,对46例孕47～65 d高危孕妇的绒毛间期细胞进行FISH检测,同时行常规染色体核型分析平行诊断.结果与染色体核型分析结果一致的染色体数目正常43例,异常3例(47,XY 21;47,XY 18和45,X).3例异常核型胚胎经治疗性流产后再分别对其绒毛行常规染色体核型分析,结果与产前诊断相符.结论FISH技术用于产前诊断孕早期胚胎染色体数目异常具有快速、准确等优点.     

早期妊娠自然流产患者的超声表现与绒毛染色体核型分析

【目的】 探讨早期妊娠自然流产患者的不同超声表现与绒毛染色体异常的关系?【方法】 2008年1月至2011年12月,在我院诊治的早期自然流产患者,流产前有定期B超和人绒毛膜促性腺激素(HCG)测定,流产后成功行绒毛细胞培养和染色体核型分析者共183例?根据流产前的B超分为有胎心组和无胎心组,比较两组的染色体核型分析结果有无差异?【结果】 183例绒毛染色体核型分析中,检出异常核型109例(59.6%),正常核型74例(40.4%)?有胎心组102例,异常染色体发生率61.8%,无胎心组81例,异常染色体发生率56.8%,两组比较无统计学差异(P > 0.05)?有胎心组中最常见的异常染色体为(45,X)?三倍体和16三体,而无胎心组中最常见的异常染色体为16三体,无1例(45,X)和三倍体?有胎心组中可存活常染色体三体(21三体?18三体?13三体)?(45,X)?三倍体29例,占异常染色体的(46.0%),明显高于无胎心组,仅3例,占异常染色体的6.5%,(P < 0.05)?在有胎心组中,可存活常染色体三体(21三体?18三体?13三体)?(45,X)?三倍体的胚芽长度平均(17.7 ± 6.4)mm,明显大于其他异常染色体的胚芽长度(8.8 ± 5.3)mm (P < 0.05)和正常染色体的胚芽长度(11.1 ± 8.4) mm(P < 0.05)?【结论】 绒毛染色体异常是早期自然流产的主要原因,可存活常染色体三体(21三体?18三体?13三体)?(45,X)?三倍体多发生于已有胎心的自然流产者?     

组织蛋白酶B和L在早期妊娠绒毛及蜕膜中的表达

目的 观察组织蛋白酶B（cathepsin B，CB）和L（cathepsin L，CL）在人早期妊娠绒毛及蜕膜中的表达．探讨它们在胚胎着床及发育过程中的作用。方法 研究组包括：正常早期妊娠行人工流产者30例、早期自然流产者25例、正常分泌期子宫内膜lO例和葡萄胎15例。应用免疫组织化学法检测CB和CL的表达。结果 CB、CL主要阳性表达于正常早期妊娠滋养层细胞和蜕膜细胞：CB在正常分泌期内膜．正常早期妊娠蜕膜、自然流产蜕膜中的阳性率表达分别为10％（1／10）、83.3％（25／30）、32．0％（8／25）；CL则分别为0、63.3％（19／30）、32．0％（8／25）；CB和CL在3组间的表达有显著性差异（P〈0．05）。CB在早期妊娠绒毛、自然流产绒毛、葡萄胎组织中表达强阳性率分别为10．0％（3／30）、0.66．7％（10／15）；CL则分别为26．7％（8／30）、4．0％（1／25）、80．0％（12／15）。CB和CL在3组间的表达有显著性差异（P〈0．05）。结论 CB和CL在正常和异常妊娠蜕膜和绒毛中的表达差异提示它们胚胎着床过程中可能起着很重要的作用。     

复发性流产与夫妻双方绒毛血管内皮生长因子基因多态性的关系

【目的】探讨复发性自然流产(RSA)与中国南方夫妻双方及绒毛血管内皮生长因子基因多态性(-1154G > A)的关系【方法】采用等位基因特异性扩增-聚合酶链反应方法,检测271对RSA夫妇和250名女性对照?200名男性对照,60例复发自然流产绒毛及人工流产绒毛的VEGF基因-1154G > A多态位点基因型频率分布情况.【结果】在女方RSA组和对照组,VEGF基因-1154G/A的三种基因型频率(G/G?G/A?A/A)分布不相同,差异有统计学意义(P = 0.012);女方RSA组和对照组等位基因频率(G?A)分别是74.7%?25.3%和82.2%?17.8%,女方携带A等位基因发生RSA的风险大于携带G等位基因(P = 0.003,OR = 1.562,95% CI = 1.157-2.109)在男方RSA组和对照组,VEGF基因-1154G/A的三种基因型频率及等位基因频率分布差异均无统计学意义(P分别为0.837?0.572)?在留取的绒毛RSA组和人工流产组,VEGF基因-1154G/A的三种基因型频率及等位基因频率分布差异均无统计学意义(P分别为0.708?0.757),【结论】女方VEGF基因-1154G/A多态与中国南方复发性自然流产的发病风险相关,携带A等位基因增加复发性自然流产的发生,男方及绒毛VEGF基因-1154G/A多态与中国南方复发性自然流产的无相关性,由此推测,与胎儿VEGF-1154G/A多态性相比,母方VEGF-1154G/A多态性与RSA的关系可能更密切     

Prenatal fetal karyotyping and maternal serum alpha-fetoprotein screening

Prenatal karyotyping was undertaken in 569 consecutive amniotic fluid samples where the indication for amniocentesis was two sequential raised maternal serum alpha-fetoprotein concentrations. In 475 successful cultures five chromosome abnormalities were found--four constitutional anomalies (47,XXY; 47,XYY; an inherited inv(8) (p23q11); and a de-novo translocation t(6;7) (p11;p22) and a culture-derived anomaly (trisomy 2) found in amniotic fluid cells but not in the fetus aborted because it had spina bifida. Of the pregnancies complicated by constitutional abnormalities, only the pregnancy in which the de-novo translocation was detected was terminated. No chromosome abnormalities were detected in the 17 pregnancies which miscarried after amniocentesis. These results provide little justification for including fetal karyotyping as an essential part of maternal serum alpha-fetoprotein screening programmes.     

自然流产绒毛与蜕膜组织中细胞凋亡的研究

目的研究细胞凋亡在自然流产患者绒毛和蜕膜组织中的表达及其在自然流产中的作用。方法应用TdT介导的dUTP缺口原位末端标记技术（terminal deoxvnucleotidvl transferase-mediated dUTP-biotin nick endinglabeling,TUN-EL）检测40例早孕自然流产病例（研究组）和40例早孕人工流产病例（对照组）绒毛和蜕膜组织中细胞凋亡情况。结果凋亡指数在自然流产绒毛细胞滋养层细胞、合体滋养层细胞和蜕膜中分别为（24．54±3．21）％、（40．25±5．27）％和（56．14±4．56）％,早孕人工流产分别为（10．31±1．25）％、（15．65±5．45）％和（10．54±1．82）％,两组间差异有高度显著性（P〈0．01）。结论自然流产患者绒毛和蜕膜组织中细胞凋亡显著增加,其在自然流产的发生发展中起一定的作用。     

自然流产患者的细胞遗传学分析

目的：探讨流产患者染色体异常的核型分布以及染色体平衡易位以生育的影响。方法：9258例自然流产患者进行染色体核型分析。采用淋巴细胞培养，常规收获制处，G显带处理。必须要加做C带、N带分析。结果：本组患者染色的异常率为2．72％，男性和女性分别为2．12％和3．32％，两者差异有显著性（P＜0．01），流产超过3次的患者，其染色体异常率显著性增加（P＜0．01）。在252例异常核型中，平衡易位，数目异常、倒位、缺失和标记染色体分别占81．0％、13．1％、4．0％、0．4％和0．8％，130例平衡易位携者有473次妊娠，流产占90．1％，死胎，分娩异常后代，分娩正常后代，分娩易位后代分别为4．7％、4．0％、1．1％、0．2％，结论：流产患者中，女性染色体异常率高于男性，流产超过3次的患者，其染色体异常显著增加，平衡易位是流产患者主要的异常核型，其妊娠结局以流产为主，应进行遗传咨询和产前诊断。     

自然流产病例的绒毛与蜕膜细胞增殖和细胞凋亡的研究

目的　从细胞增殖与细胞凋亡的角度探讨自然流产的发生机制。方法　对 30例正常早孕吸宫流产病例和 30例自然流产病例的绒毛与蜕膜组织进行研究。常规光镜观察细胞形态学变化 ;免疫组织化学S P法检测增殖细胞核抗原 (PCNA)在细胞中的表达 ;用DNA缺口原位未端标记技术 (TUNEL)检测细胞凋亡 ;免疫组化S P法检测bcl 2 bax基因蛋白表达率比值。结果　自然流产绒毛组织形态学结构均出现程度不同的退行性改变 ,绒毛的细胞滋养细胞及蜕膜中增殖指数 (PI)较正常早孕时明显减少 (P <0 0 1) ,而合体滋养细胞中从未发生过增殖 ,PI为 0 ;与此相反自然流产绒毛的细胞滋养细胞、合体滋养细胞及蜕膜中凋亡指数 (AI)较正常早孕时明显增多 (P <0 0 5 ,P <0 0 1) ,同时 ,早孕流产的绒毛及蜕膜中促 /抑凋亡基因bcl 2 bax表达率的比值也较正常早孕时下降 (P <0 0 5 ,P <0 0 1) ,结果与细胞凋亡的发生一致。结论　细胞增殖和凋亡可能在孕卵发育过程中起着重要作用 ,绒毛和蜕膜组织中大量的细胞凋亡是其形态学退变的根本 ,并进而阻碍了孕卵的发育。     

绒毛直接制备染色体的改良方法

本文针对绒毛直接制备染色体中存在分裂和质量不稳定的情况，而增加固定液作用时间。将低渗后的绒毛细胞换固定液后置冰箱（±4℃）过夜，并在60％冰乙酸解离绒毛细胞后再换固定液作用3小时或置冰箱过夜。结果所获得染色体形态、分散较以前改进。并可获得较高质量的分裂相。21例计数结果每例可分析的染色体占分裂相数的40.93％。     

Effect of chorionic villus sampling and early pregnancy counselling on uptake of prenatal diagnosis

An early pregnancy counselling clinic was introduced to improve the uptake of prenatal diagnosis and to offer chorionic villus sampling to women aged 38 and over by their expected date of delivery. Ninety eight (62%) unselected older mothers were seen before 11 weeks' gestation, and 23 (32%) of those with viable pregnancies elected to undergo chorionic villus sampling compared with 38 (52%) electing amniocentesis. A quarter of the patients booking before 11 weeks had a miscarriage. Because of the future potential demand for chorionic villus sampling counselling during pregnancy and referral of eligible patients should occur as early as possible.     

不孕症和自然流产患者染色体核型异常分析

目的：分析评价不孕症和自然流产患者染色体核型分布的关系。方法整群收集该院2012年1月—2015年12月行外周血染色体检查的不孕症和自然流产患者416例进行G显带染色体核型分析,发现异染色区异常病例进行C显带确认,发现随体及随体柄异常进行N显带确认。结果在对该院就诊的不孕症和自然流产患者核型分析检查的416例样品中,共检出的51例染色体异常核型和2例性发育异常病例,总异常率为12.26％。51例染色体异常核型中常染色体结构异常有34例;异染色区多态性9例;性染色体结构异常4例。染色体数目异常4例,其中1例为嵌合型数目异常。结论染色体核型异常与不孕症和自然流产密切相关,也是出生异常胎儿的重要原因,为达到降低出生人口缺陷率优生优育的目标应进行必要的遗传学咨询、产前筛查、产前诊断。     

Prenatal cytogenetic diagnosis study of 2782 cases of high-risk pregnant women

Background Prenatal diagnoses are extremely advantageous for pregnant women with high-risk indicators and can help prevent the birth of malformed infants. However, no large-scale statistical study analyzing the correlation between fetal chromosome disorders and abnormal indicators during pregnancy has been done in China. The objectives of this study were to diagnose and analyze fetal chromosome abnormalities, determine the feasibility of the various prenatal test methods and establish diagnostic guidelines for the early, middle, and late trimesters. Methods From January 2004 to May 2009, 2782 pregnant women at high-risk underwent prenatal diagnoses. Categorized data expressed as either actual counts or percentages were analyzed by the chi-square or Fisher＇s exact test. Chorionic villus sampling was performed in the early-trimester （10-12 weeks of gestation）, amniocentesis in mid-trimester （16-28 weeks of gestation）, and umbilical cord blood collection in mid- or late-trimester （16-37 weeks of gestation）. In 51 cases either autopsy samples from intrauterine fetal deaths or placental tissues from aborted fetuses were tested. Results Chromosomal abnormalities were observed in 3.99% （111/2782） of the samples. Overall, the success rate of cytogenetic analysis for high-risk pregnancy groups was 98.17% （2731/2782）. It was significantly less successful when used to analyze data from the chorionic villus sampling compared with that from amniocentesis and umbilical cord blood （P=-0.000）. Abnormal chromosome carriers had the highest percentage of abnormal chromosomes （67.86%） when compared with chromosomal abnormalities in patients with ultra-sonographic ＂soft markers＂ （11.81%）, advanced maternal age （4.51%） and those who had positive serum screening results （P=-0.000）. Conclusions Invasive prenatal diagnostic techniques are feasible tools for confirming fetal chromosomal abnormalities. Abnormal chromosomes detected in one of the parents carrying abnormal chromosome, ultrasound soft markers, advanced maternal age or positive serum screening results were associated with a higher frequency of fetal genetic diseases.     

早期自然流产胎儿绒毛细胞培养及染色体核型分析

目的 探讨早期自然流的原因及流产胎儿绒毛染色体核型检查的意义.方法 应用长期培养法对60例早期自然流产的胎儿绒毛组织进行绒毛细胞培养,并对培养成功的绒毛细胞制备染色体及核型分析.结果 60例自然流产的绒毛组织中绒毛细胞培养成功56例(93.3%),56例中检出异常核型32例(57.1%),其中常染色体三体16例、多倍体...     

基质金属蛋白酶诱导因子在先兆流产绒毛及蜕膜组织中的表达

目的探讨先兆流产时绒毛及蜕膜组织中细胞外基质金属蛋白酶诱导因子（EMMPRIN）的表达情况。方法选取30例妊娠早期发生先兆流产的妇女,30例自愿行负压吸引术终止妊娠的早期正常妊娠妇女,在获取绒毛及蜕膜组织后应用免疫组织化学技术对EMMPRIN进行检测。结果（1）先兆流产组绒毛表达的EMMPRIN强于正常妊娠组,且增强部位为细胞滋养层柱细胞与合体滋养细胞。（2）EMMPRIN在先兆流产组、正常妊娠组蜕膜组织中均呈阳性表达,且二者表达的EMMPRIN无明显差异。结论发生先兆流产时合体滋养细胞、细胞滋养层柱所表达的EMMPRIN增强可能通过上调绒毛及蜕膜组织中基质金属蛋白酶（MMPs）的水平,降解细胞外基质（ECM）,使绒毛剥离、蜕膜组织剥脱。     

Assessment of the early risks of chorionic villus sampling.

A study was carried out to develop proficiency in performing chorionic villus sampling (CVS) and to determine whether the risk for miscarriage was so high as to preclude a randomized clinical trial comparing CVS with amniocentesis. A total of 202 women who had decided to have induced abortions volunteered to either undergo CVS (101 women) or be a control (101). There were no differences between the two groups in factors that may affect the rate of miscarriage. CVS was performed an average of 9.8 days before abortion. The rate of fetal loss was significantly higher in the CVS group (p = 0.009). An analysis of the results as a function of the physician's experience over time showed that there were distinct learning phases. It may take longer than is generally recognized to acquire the expertise necessary to perform CVS with the lowest risk possible. Caution should be exercised before diagnostic CVS is offered to women who plan to continue their pregnancies.     

Rapid aneuploidy testing (knowing less) versus traditional karyotyping (knowing more) for advanced maternal age: what would be missed, who should decide?

OBJECTIVES: The application of rapid aneuploidy testing as a stand-alone approach in prenatal diagnosis is much debated. The major criticism of this targeted approach is that it will not detect other chromosomal abnormalities that will be picked up by traditional karyotyping. This study aimed to study the nature of such chromosomal abnormalities and whether parents would choose to terminate affected pregnancies. DESIGN: Retrospective study on a cytogenetic database. SETTING: Eight public hospitals in Hong Kong. PARTICIPANTS: The karyotype results of 19 517 amniotic fluid cultures performed for advanced maternal age (>or=35 years) from 1997 to 2002 were classified according to whether they were detectable by rapid aneuploidy testing. The outcomes of pregnancies with abnormal karyotypes were reviewed from patient records. RESULTS: In all, 333 (1.7%) amniotic fluid cultures yielded abnormal karyotypes; 175 (52.6%) of these were detected by rapid aneuploidy testing, and included trisomy 21 (n=94, 28.2%), trisomy 18 or 13 (n=21, 6.3%), and sex chromosome abnormalities (n=60, 18.0%). The other 158 (47.4%) chromosomal abnormalities were not detectable by rapid aneuploidy testing, of which 63 (18.9%) were regarded to be of potential clinical significance and 95 (28.5%) of no clinical significance. Pregnancy outcomes in 327/333 (98.2%) of these patients were retrieved. In total, 143 (42.9%) of these pregnancies were terminated: 93/94 (98.9%) for trisomy 21, 20/21 (95.2%) for trisomy 18 or 13, 19/60 (31.7%) for sex chromosome abnormalities, and 11/63 (17.5%) for other chromosomal abnormalities with potential clinical significance. There were no terminations in the 95 pregnancies in which karyotyping results were regarded to be of no clinical significance. CONCLUSIONS: 'Knowing less' by the rapid aneuploidy stand-alone testing could miss about half of all chromosomal abnormalities detectable by amniocentesis performed for advanced maternal age. Findings from two fifths of the latter were of potential clinical significance, and the parents chose to terminate one out of six of the corresponding pregnancies. If both techniques are available, parents could have enhanced autonomy to choose.     

GM-CSF及其受体与早期自然流产的关系研究

目的：探讨母胎界面局部细胞因子GM-CSF及其受体与早期自然流产的发生的关系．方法采集2009年8月至2011年12月间来云南省第三人民医院进行人工流产及自然流产病例的绒毛及脱膜组织各30例,用放射免疫法检测2组外周血中绒毛膜促性腺激素（HCG）含量,利用免疫组织化学和Western blot检测绒毛及脱膜组织中巨噬细胞集落刺激因子（GM-CSF）及其受体（GM-CSFR）表达情况．结果自然流产组HCG值与人工流产组相比明显下降（P〈0.05）．绒毛及脱膜组织中GM-CSF和GM-CSFR 2组均有表达．与人工流产组相比,绒毛组织中,自然流产组的 GM-CSFR增加（P〈0.05）,GM-CSF的表达上调,但无统计学意义（＞0.05）．脱膜组织中,自然流产组的GM-CSF和GM-CSFR均增加（P〈0.05）．结论在脱膜组织中,适量的GM-CSF和GM-CSFR浓度维持妊娠的继续,浓度过高则不利于妊娠,可能是自然流产的原因之一．