单核苷酸多态性微阵列芯片在分析流产或死胎染色体中的应用

目的:探讨单核苷酸多态性微阵列芯片在流产或死胎染色体中的应用价值,同时研究流产频率与母体年龄和流产物染色体异常的关系.方法:釆用单核苷酸多态性微阵列芯片对流产绒毛、死胎组织和引产羊水进行遗传学检测.结果:673例样本检测失败9例,检测成功率98.7％,664例样本中共检出388例(58.4％)染色体异常,其中非整倍体异...     

拷贝数变异测序在染色体易位夫妇植入前遗传学诊断中的临床应用

目的探讨拷贝数变异测序(copy number variation sequencing, CNV-seq)用于染色体易位夫妇胚胎植入前诊断的应用价值。 方法回顾性分析2017年1月至2018年12月,在广东省妇幼保健院生殖健康与不孕症科进行植入前遗传学诊断(preimplantation genetic diagnosis,PGD)的211对染色体易位夫妇患者的临床病例。使用CNV-seq对胚胎染色体进行检测,并对患者一般信息和PGD结果进行分析。 结果1210个胚胎中,被检出837个(79.2%)胚胎存在染色体异常,373个(30.8%)胚胎为整倍体。在241个PGD周期中,68个(27.6%)周期所有胚胎均存在染色体异常,178个(72.4%)周期至少含有一个整倍体胚胎。在176个移植周期中,130个(73.9%)确定临床妊娠,已出生46个健康婴儿,12例发生早期流产。 结论CNV-seq可准确地区分胚胎染色体是否存在异常,避免因胚胎含有染色体异常而被移植,是一种可靠而准确的PGD技术。     

测序技术检测流产物拷贝数目变异的应用

目的:采用测序技术检测拷贝数变异,探讨测序技术在染色体疾病上的应用和拷贝数变异在流产物检测中的应用价值。方法:选择2013年7月至2014年4月在贵阳医学院附属医院贵州省产前诊断中心就诊孕妇中胚胎停止发育的50例患者,孕周为7~23周,留取流产物,提取DNA,制备文库,高通量测序,对流产物DNA进行测序分析,得出流产物的拷贝数变异。同时对孕妇和流产物做染色体核型分析。结果:50例孕妇的流产物染色体基因拷贝数变异检测,除1例为母源性污染,未得出结果外,测序检出率78%,染色体核型检出率为28%。结论:测序技术检测流产物的检出阳性率高于染色体核型检出率,流产物染色体核型能检出的整倍体改变、非平衡易位改变,测序技术均能检出,且在染色体微缺失微重复检出上优于染色体核型检出,推荐临床上使用对流产物进行测序技术检测查找流产原因。     

86例稽留流产组织的染色体检测

目的探讨胚胎稽留流产与染色体异常之间的关系。方法采用着丝粒、专一序列探针(13、16、18、21、22、X、Y)对稽留流产绒毛组织和胎儿组织进行FISH检测。结果86例稽留流产组织采用FISH技术检测,共检测出44例染色体异常,异常检出率为51.16%。其中79例流产绒毛组织检出43例染色体异常,分别为2例13-三体,9例16-三体,5例18-三体,2例21-三体,9例22-三体,5例性染色体单体,1例XXY,1例XXX,7例三倍体,2例四倍体;7例胎儿组织中发现1例异常,为18-三体。流产组织染色体异常组孕妇年龄和正常组比较及流产组织常染色体异常组胎儿性别与正常组比较,差异均无统计学意义(P0.05)。结论染色体异常是导致胚胎稽留流产发生的重要原因,因此对流产胚胎进行染色体检测,不仅可以了解本次流产的原因,而且对下一次受孕有很大的指导意义。     

细菌人工染色体标记-微球鉴别/分离法在产前诊断中的临床应用价值

目的:探讨细菌人工染色体标记-微球鉴别/分离法(BoBs)在产前诊断中的临床应用价值。方法:对2015年1月至2018年4月空军军医大学第一附属医院4882例有产前诊断指征的孕妇羊水细胞行染色体核型分析和BoBs检测,检测结果进行比较分析。结果:4882例羊水中共检出胎儿染色体异常289例,异常检出率5.92%。其中染色体核型分析检出271例,BoBs检出266例。不同产前诊断指征下,无创产前检测高风险组的染色体异常检出率最高,占56.00%。289例异常核型中,染色体非整倍体239例,BoBs检测结果与染色体核型分析结果吻合;染色体微缺失/微重复综合征21例,染色体核型分析仅检出3例;性染色体嵌合11例,BoBs检出6例;染色体结构异常18例,BoBs均未检出。结论:BoBs技术是一种可靠的检测技术,可以全面快速检测胎儿染色体非整倍体及9种常见的微缺失/微重复综合征,与染色体核型分析联合可以提高产前诊断的效率及准确性,具有较高的临床应用价值。     

染色体微阵列分析技术在复发性自然流产遗传学诊断中的应用

目的:探讨染色体微阵列分析(CMA)在复发性自然流产(RM)遗传学诊断中的应用价值,研究既往流产次数与胚胎染色体异常率的关系以及致病性微缺失/微重复与RM的相关性。方法:选取2011年8月至2021年9月在南京医科大学附属妇产医院就诊的1355例RM病例,取流产绒毛行CMA检测。结果:排除14例(1.0%,14/1355)重度母体细胞污染样本,共1341例病例纳入研究。1341例RM病例中,共检出致病性染色体异常813例(60.6%),其中异倍体597例(44.5%)、多倍体113例(8.4%)、大片段结构异常67例(5.0%)、致病性微缺失/微重复25例(1.9%)、单亲二体(UPD)11例(0.8%)。孕妇流产2次、3次和≥4次组病例的染色体异常率分别为62.6%、56.5%和51.9%,流产2次组病例的染色体异常率显著高于流产3次、≥4次组(P<0.05)。本研究发现5个可能与RM发生相关的复发性(n≥2)致病性微缺失/微重复(15q11.2、19p13.3、22q13.2q13.33微缺失和17p13.3、22q11.1q11.21微重复),其中22q13.2q13.33...     

61例孕早期稽留流产绒毛应用高通量测序技术染色体检测结果分析

目的:应用高通量测序技术检测稽留流产胚胎绒毛组织染色体,探讨其在早期自然流产遗传学诊断中的应用价值。方法:高通量测序检测61例稽留流产孕妇(孕5～13周)的绒毛组织,统计染色体异常的类型及所占比率。结果:61例稽留流产患者绒毛组织中,染色体异常38例。染色体数目异常28例,其中三体型占34.4%(20/61),X单体占9.8%(6/61),嵌合体占3.3%(2/61);染色体结构异常10例,占16.4%(10/61)。孕妇年龄≥35岁组和35岁组的绒毛染色体异常率分别为76.9%和51.4%,两组比较差异有统计学意义(P0.05)。既往有不良孕产史及无不良孕产史者的绒毛染色体异常率分别为73.5%和44.4%,两组比较差异有统计学意义(P0.05)。结论:染色体异常是早期稽留流产的重要原因,高龄及不良孕产史可能增加胚胎染色体异常的风险。高通量测序技术应用于绒毛染色体检测对于分析稽留流产的原因具有重要意义,有助于明确稽留流产病因,在提供合理的优生遗传咨询并指导下一次良好妊娠有积极的应用价值。     

细菌人工染色体标记-微球鉴别/分离法对1973份羊水的快速产前诊断

目的探讨细菌人工染色体标记-微球鉴别/分离法(bacterial artificial chromosomes-on-beads,BoBs)在产前诊断中的应用价值。方法对2015年1月至2016年6月西京医院产前诊断中心1973例有产前诊断指征的孕妇羊水细胞进行BoBs检测和染色体核型分析。结果 1973例羊水中,共发现异常核型150例,其中染色体核型分析检出145例,BoBs检出133例。不同产前诊断指征下,无创DNA产前检测(NIPT)高风险组的染色体异常检出率最高,占该指征的81.82%。150例异常核型中,BoBs和核型分析均检出染色体非整倍体124例,占异常核型的82.67%;BoBs检出染色体微缺失/微重复综合征5例,核型分析结果均未见异常;染色体核型分析检出性染色体嵌合7例,BoBs检出4例;染色体核型分析检出结构异常14例,BoBs结果未见异常。结论 BoBs技术可以快速检测染色体非整倍体和基因微缺失/微重复,联合染色体核型、基因芯片(chromosomal microarray analysis,CMA)及荧光原位杂交技术(fluorescence in situ hybridization,FISH)可对染色体结构异常和低嵌合正确检出,大大提高了产前诊断的效率与准确性。     

高通量连接探针扩增技术联合STR分型在孕早期稽留流产中的应用

目的:探讨高通量连接探针扩增(HLPA)技术联合短串联重复序列(STR)技术在流产病因学研究中的临床应用价值。方法:收集孕7～13⁺⁶周稽留流产样本283例，先行STR检测，再分别采用HLPA或染色体微阵列芯片(CMA)技术进行遗传学检测。结果:本组样本检测总成功率为98.94%(280/283),3例STR检测不通过，未进行后续检测，共检出染色体异常159例，异常检出率为56.79%(159/280),其中染色体数目异常94.34%(150/159),染色体结构畸变5.03%(8/159),单亲二倍体0.63%(1/159)。HLPA成功检测140例，染色体异常56.43%(79/140);CMA成功检测140例，染色体异常57.14%(80/140),两者差异无统计学意义。结论:HLPA+STR技术和CMA同样具有准确度高的优点。STR分型可排除母体污染，同时对HLPA在多倍体以及单亲二倍体的检测方面起到补充作用，两者联合应用可成为孕早期胚胎停止发育遗传学检测方法。     

高通量测序技术检测自然流产绒毛样本的染色体拷贝数变异情况

目的探讨高通量测序(NGS)技术在检测自然流产绒毛样本的遗传学分析准确性和异常结果检出率中的应用价值。方法选取早期自然流产患者的绒毛组织作为研究对象,采用NGS方法对流产绒毛组织的染色体拷贝数变异情况进行检测。同时,我们采用比较基因组杂交芯片(a-CGH)法进行验证,以确保NGS检测结果的准确性。结果本研究共纳入256例绒毛样本,检测成功率100%。染色体异常共计145例(56.64%),染色体未见明显异常共计111例(43.36%)。染色体微缺失/微重复共34例(13.28%)。在染色体异常结果中,非整倍体128例(50%),其中三体共计94例(36.72%),主要涉及22号和16号染色体,占三体总例数的50%;单体共24例(9.37%),Turner综合征为22例(91.7%);四体共10例(3.91%)。染色体结构异常共17例(6.64%)。随机选取的5例样本同时采用a-CGH芯片检测,结果与高通量测序结果一致。结论 NGS技术是一项敏感、高效的遗传学检测手段,可有助于明确自然流产的遗传学因素,以期指导下次妊娠。     

早期自然流产绒毛组织SNP-array遗传学诊断研究

目的:初步探讨单核苷酸多态性阵列(SNP-array)在早期流产绒毛遗传学诊断中的临床应用价值。方法:选取临床诊断为早期自然流产的82例患者,刮宫术后获取绒毛组织,行常规绒毛细胞培养G显带核型分析,并同时提取绒毛组织DNA进行SNP-array检测,比较两者的检测结果。结果:常规绒毛细胞培养G显带核型诊断成功率87.8%(72/82),SNP-array诊断成功率为100%(82/82)。G显带分析获得结果 72例,核型正常35例,核型异常37例,异常率51.4%(37/72)。82例SNP-array分析结果中,核型正常30例,核型异常52例,异常率63.4%(52/82)。G显带分析失败的10例标本中,SNP-array检出6例异常;G显带与SNP-array结果不符的12例中,包括2例全基因组单亲二倍体(uniparental disomy,UPD),2例是部分染色体UPD。结论:SNP-array技术具有高准确性、高通量、快速检测等优点,在自然流产绒毛遗传学分析中具有较强的临床应用价值。     

Routine histopathologic analysis of product of conception following first-trimester spontaneous miscarriages

AIM: To evaluate the histopathologic findings relating to tissue samples collected at surgical uterine evacuation in first-trimester spontaneous miscarriages. METHODS: In this retrospective study, histopathologic diagnosis of the tissue samples obtained via surgical uterine evacuation in patients who were admitted to the Early Pregnancy Clinic in a 12-month period with the diagnosis of incomplete miscarriage (n = 970), missed miscarriage (n = 406) and anembryonic miscarriage (n = 230) in the first trimester was recorded and compared with the presurgery diagnosis. RESULTS: Uterine evacuation was performed in cases of incomplete miscarriage (n = 970, 60.4%), missed miscarriage (n = 406, 25.2%) and anembryonic miscarriage (n = 230, 14.3%). Histopathologic examination revealed the product of conception in 1119 patients (69.7%), while partial hydatidiform mole was diagnosed in 33 patients (2.1%). Complete hydatidiform mole was detected in only seven cases (0.43%). Exaggerated placental site and placental site trophoblastic nodule was detected in two cases (0.12%). Decidual tissue without chorionic villi was reported in 272 patients (16.9%), raising the suspicion of presence of other pathology. CONCLUSIONS: By routine histopathologic assessment of products of first-trimester spontaneous miscarriages, important pathologies such as molar pregnancy and placental trophoblastic disease can be diagnosed. Histopathological assessment has great value in the identification of an ectopic pregnancy or infection when compared with clinical and laboratory findings.     

Chorionic villus sampling for first-trimester prenatal diagnosis: Northwestern University program

We present our initial experience in developing a chorionic villus sampling program at Northwestern University. In phase 1, we performed chorionic villus sampling in 58 patients prior to elective first-trimester abortion, assessing the reliability and reproducibility of obtaining adequate villus samples and performing cytogenetic analysis by means of both the direct and culture methods. Specimens were categorized according to quality: class I, multiple identifiable villi (n = 20); class II, few villi or villi mixed with decidua (n = 15); class III, no villi (n = 23). There was a positive trend between operator experience, amount of villi obtained, and quality of cytogenetic preparations. In March, 1984, we received Institutional Review Board approval to perform chorionic villus sampling in continuing pregnancies (phase 2). Among the first 20 cases we found two abnormalities (47,XY, + 13; 45,X). The remaining 18 pregnancies were continuing. Recommendations are made for developing a chorionic villus sampling program.     

Chromosomal abnormalities in products of conception of first-trimester miscarriages detected by conventional cytogenetic analysis: a review of 1000 cases

Purpose

The purpose of this study is to perform a retrospective analysis of types and frequencies of chromosomal abnormalities detected by conventional cytogenetic studies in first-trimester miscarriages after spontaneous conception and IVF.

Methods

Standard cytogenetic analysis of GTG-banded chromosomes obtained from products of conception (POCs): semi-direct and short-term cultured chorionic villi or long-term cultured fetal mesodermal cells.

Results

50.1% of first-trimester miscarriages in the studied group had chromosomal abnormalities: 59.7% of trisomies, 22% of poliploidies, 7.5% of monosomies, 7% of unbalanced structural abnormalities, and 3.8% of multiple aneuploidies. An increase in the frequency of chromosomally abnormal miscarriages was observed in the group of women above 40 when compared to groups of women under 35 (P < 0.05). No difference in frequencies and types of chromosomal abnormalities in POCs of miscarriages after ICSI and spontaneous conception was observed.

Conclusions

Approximately, 50% of first-trimester miscarriages have chromosomal abnormalities which can be detected by conventional cytogenetic analysis. The presence of chromosomal abnormality may explain the cause of miscarriage, improving the reproductive counseling and planning.

     

Genetic aspects of miscarriage.

Fetal chromosome abnormalities account for about 50% of first-trimester pregnancy losses. Most of these abnormalities are numerical abnormalities (86%) and a low percentage is caused by structural abnormalities (6%) or other genetic mechanisms, including chromosome mosaicism (8%). The recurrence risk of numerical abnormalities is low, so karyotyping of fetal material in case of a miscarriage does not seem worthwhile in daily practice.Half of the structural abnormalities may be inherited from a parent carrying a balanced chromosome translocation or inversion. Parental carriership is found in 4-6% of the couples with recurrent miscarriage. In case of parental carriership of a balanced structural chromosome abnormality, a next pregnancy may result in a child with an unbalanced structural chromosome abnormality. This child can have multiple congenital malformations and/or a mental handicap. Prenatal diagnosis is therefore recommended.Conventional laboratory techniques, such as tissue culturing and karyotyping, or (semi-)direct chromosome technique of chorionic villi, and the recently developed laboratory techniques such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), are described successively.Until now, not enough evidence has been available about the role of other genetic mechanisms, such as single-gene abnormalities, uniparental disomy, genomic imprinting, multifactorial disorders and skewed X chromosome, in the occurrence of miscarriages.     

Chorionic villi sampling: clinical experience, immediate complications, and patient attitudes

To develop chorionic villi sampling as a procedure for prenatal diagnosis, a pilot study was undertaken to perfect the obstetric and laboratory techniques, to evaluate our success with the procedure in continuing pregnancies, and to assess the attitudes of potential users of the procedure. Women about to have elective first-trimester abortions for nongenetic reasons were enrolled in the first phase of the study. Of the patients with a positive pregnancy test, 12.4% were found to have a nonviable pregnancy on ultrasound examination. Samples adequate for cytogenetic analysis were obtained in 130 of the 155 remaining cases, and the success rate was 93% in the 100 most recent cases. Direct cytogenetic analysis was undertaken in those cases successfully sampled, and karyotypes could be prepared in 97%. Immediate complications occurred in 5% of the pregnancies. Eight women at risk of bearing a child with a genetic defect had diagnostic chorionic villi sampling. Cytogenetic analysis was performed successfully on all of them. One had an induced abortion following the procedure because of the fetal diagnosis (a male with a 50% risk of Duchenne's muscular dystrophy). The other pregnancies are continuing uneventfully at 22 to 35 weeks' gestation. Finally, from preliminary analysis of our survey of potential users it appears that women 35 years old or over would prefer chorionic villi sampling to amniocentesis if the risks of the sampling were known to be low.     

First-trimester prenatal diagnosis performed on pregnant women with fetal ultrasound abnormalities: The reliability of interphase fluorescence in situ hybridization (FISH) on mesenchymal core for the main aneuploidies

Objectives

To examine the reliability of interphase FISH analysis of the main aneuploidies performed on mesenchymal core when prenatal diagnosis was performed on pregnant women with first-trimester fetal abnormalities on ultrasound.

Study design

386 first-trimester prenatal examinations were investigated from chorionic villus samplings for increased nuchal translucencies or other fetal ultrasound abnormalities. Interphase fluorescence in situ hybridization (FISH) for the main aneuploidies (trisomies 13, 18, 21 and gonosomal aneuploidies) was performed on the mesenchymal core of villi. Molecular cytogenetic results were always complemented by conventional cytogenetic results on long-term cultured villi (LTC-villi). Short-term cultured villi (STC-villi) preparations were retrospectively performed only when a chromosomal abnormality was observed with interphase FISH and/or LTC-villi.

Results

88 chromosomal abnormalities (88/386 = 22.8% of first-trimester diagnoses) which could discuss subsequent abortions were observed after LTC-villi preparations. All cases possibly detectable by interphase FISH were detected. Thus, 85 aneuploidies (85/386 = 22.0% of first-trimester diagnoses; 85/88 = 96.6% of chromosomal abnormalities) were detected by interphase FISH, allowing early abortion by curettage before week 14 amenorrhea. No discrepancy occurred between interphase FISH and LTC-villi results for the aneuploidies studied. Three false-negative results (3/386 = 0.77% of first-trimester diagnoses; 3/88 = 3.41% of chromosomal abnormalities) were observed with STC-villi.

Conclusion

We observed a high rate of false-negative results on cytotrophoblast cells. Conversely, interphase FISH of the main aneuploidies on the mesenchymal core provided rapid and reliable results, and therefore should be preferred to STC-villi in first-trimester prenatal diagnosis performed on pregnant women with fetal abnormalities on ultrasound.     

A comparison in concentration of heat shock proteins (HSP) 70 and 90 on chorionic villi of human placenta in normal pregnancies and in missed miscarriages

PURPOSE: To investigate the role of heat shock protein (HSP) on the chorionic villi of human placental cells and to compare the concentration of placental HSP70 & 90 in term deliveries and in missed miscarriages. MATERIALS AND METHODS: Fifty products of conception from women who experienced first trimester missed miscarriage and 50 placentas from women who gave birth at term were studied. An immunohistochemical investigation was carried out with which we marked the localization of heat shock proteins 70 and 90 on the syncytiotrophoblastic, cytotrophoblastic, stromal and blood vessel cells, using specific antibodies which can detect the presence of those proteins on light microscopy. We compared their expression with the normal placental tissue of term pregnancies and with material acquired from first trimester missed miscarriages. An indirect immunoperoxidase method was applied using polyclonal antibodies against HSP70 and HSP90 on formalin-fixed paraffin-embedded tissues. RESULTS: Expression of HSP90B was increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. There was a statistically significant increase of HSP90A expression in chorionic villi of first trimester missed miscarriages, concerning only the cytotrophoblast cells, compared to full-term placentas. Expression of HSP70 cognate protein was significantly increased in chorionic villi of first trimester missed miscarriages, concerning syncytiotrophoblastic cells only, compared to full-term placentas. Finally, HSP70 inducible protein was significantly increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. CONCLUSIONS: The results of the present study have sufficiently shown that there is an increase of HSP70 & 90 expression in chorionic villi of first trimester missed miscarriages compared to full-term placentas and this increase may have an important implication on the miscarriage process.     

Screening for Down syndrome: practice patterns and knowledge of obstetricians and gynecologists

OBJECTIVE: To assess obstetricians' practice patterns and knowledge regarding screening for Down syndrome. METHODS: A questionnaire was mailed to 1,105 American College of Obstetricians and Gynecologists Fellows and Junior Fellows in 2004. RESULTS: Sixty percent of questionnaires were returned. Statistical analyses were limited to the 532 practicing obstetricians. Greater than 80% felt their training and experience qualified them to counsel patients about genetic issues in pregnancy. However, 45% rated their residency training regarding prenatal diagnosis as barely adequate or nonexistent. American College of Obstetricians and Gynecologists publications were rated by 86% as an important source of information on genetic counseling. Seventy-eight percent of practitioners counsel all obstetric patients about risks for fetal aneuploidy, and 67% provide counseling for heritable genetic abnormalities. Although the majority (99%) offer second-trimester Down syndrome screening, only 55% also offer first-trimester screening for Down syndrome. Almost one half (49%) use the quad screen, and 6% offer integrated first- and second-trimester screening. The majority (88%) routinely offer amniocentesis to patients who are at elevated risk for genetic abnormalities, whereas 44% also offer chorionic villus sampling. Few (2%) perform chorionic villus sampling. CONCLUSION: Most obstetricians manage patients at risk for fetal genetic abnormalities according to American College of Obstetricians and Gynecologists educational materials. This survey identified deficiencies related to Down syndrome screening, including a limited number of practitioners performing chorionic villus sampling and physicians' own perception that training regarding genetic counseling should be improved. Educational strategies are needed to address these deficiencies before first-trimester screening programs are widely implemented. LEVEL OF EVIDENCE: III.     

(Potential) false-negative diagnoses in chorionic villi and a review of the literature

OBJECTIVES: To assess the incidence of (potential) false-negative findings of cytogenetic diagnosis in STC-villi and/or LTC-villi and to determine the best strategy for karyotyping chorionic villi in order to avoid false-negative results. METHODS: 2476 chorionic villus samples were received for prenatal cytogenetic investigations. Karyotyping was routinely performed on STC- and LTC-villi preparations by G-banding. Fluorescence in situ hybridization (FISH) analyses were performed in addition to standard chromosome analysis when necessary. Sometimes follow-up investigations like amniocentesis were performed before a definite prenatal cytogenetic result could be reported. RESULTS: In 2389/2476 (96.5%) of the cases, both STC- and LTC-villi were investigated. Normal STC- with abnormal LTC-villi results and finally an abnormal fetal karyotype were detected in ten cases (10/2389; 0.42%); in 9/10 of the cases the indication was fetal ultrasound abnormalities. Normal STC- and LTC-villi and finally an abnormal fetal karyotype were detected in two cases (2/2389; 0.08%). CONCLUSION: The most reliable technique for prenatal diagnosis after chorionic villus sampling (CVS) is the combination of the analysis of both STC- and LTC-villi to reduce the incidence of false-negative findings to a minimum. In the case of fetal ultrasound abnormalities with a small amount of villi available, the investigation of LTC-villi is recommended over that of STC-villi.