双色共变性荧光原位杂交产前诊断胎儿唐氏综合征

目的 探讨双色共变性荧光原位杂交用于非侵入性产前诊断胎儿唐氏综合征的可行性。方法 对11例孕妇外周血中的胎儿有核红细胞进行抗血型糖蛋白磁珠直接标记,再经磁激活细胞分选法富集,以Y和21号染色体专一探针对分离的胎儿有核红细胞行双色共变性荧光原位杂交,预测胎儿21号染色体倍性和性别,并用羊水染色体核型分析结果,验证预测准确性。结果 11例胎儿21号染色体倍性均正常,与羊水染色体核型分析结果相符。其中5例为男性胎儿,男性胎儿有核红细胞数量为9－65个,平均为25个,男性胎儿有核红细胞纯度为1．4％－18．8％;6例为女性胎儿,孕妇外周血中未见男性胎儿有核红细胞;性别预测结果与羊水染色体型分析结果一致。结论 双色共变性荧光原位杂交用于分析胎儿21号染色体倍性及性别,诊断胎儿唐氏综合征准确、可靠。     

Fetal cells in cervical mucus in the first trimester of pregnancy

OBJECTIVES: The aim of this study was to first evaluate the presence of fetal cells in cervical mucus samples collected in the first trimester of pregnancy and then to compare different laboratory methods for the detection of these cells. METHODS: Mucus samples were collected by using a cytobrush before termination of pregnancy (TOP) from 143 pregnant women between 7 and 12 weeks of gestation. None of the women had undergone an invasive diagnostic procedure prior to cervical mucus sampling. Samples of placental tissue were collected from each patient at TOP. Slides from each sample were first observed under an inverted microscope to detect possible sperm contamination.In the first part of our experiments, 40 mucus samples were treated with a mucolytic solution containing N-acetylcysteine (AC) and were analysed by a polymerase chain reaction (PCR) assay. The second series, consisting of 71 mucus samples, was treated with a mucolytic solution containing dithiothreitol (DTT): all 71 samples were analysed by a PCR-based assay, and an aliquot for fluorescent in situ hybridisation (FISH) analysis was also obtained from 48 out of 71 samples. In the third part of our experiments, performed on 32 mucus samples, mucus trapped on the cytobrush was directly spread on two slides for FISH analysis without any mucolytic treatment. All placental tissue samples obtained at termination were analysed by FISH for fetal sexing. RESULTS: Overall, the use of PCR-based or FISH analyses on 143 mucus samples resulted in correct sex prediction in 92/143 (64.3%) samples [20/66 (30.3%) cases from known male pregnancies and 72/77 (93.5%) cases from known female pregnancies]. In the AC group, Y-derived sequences were found in 7/23 samples (30.4%) from known male pregnancies and in 1/17 cases from known female pregnancies, with an overall correct sex prediction in 23/40 cases (57.5%). In the DTT group, Y-derived sequences could be amplified in 10/30 samples (33.3%) from known male pregnancies and in 4/41 cases from known female pregnancies, with an overall correct sex prediction in 47/71 cases (66.2%). In the DTT samples analysed by FISH, nuclei bearing XY signals were detected in 5/26 (19.2%) cases from known male pregnancies and in none from female pregnancies, the rate of correct sex prediction being 56.2% (27/48). On untreated mucus samples analysed by FISH, nuclei with XY signals were documented in 3/13 (23%) samples from male conceptuses and in none from known female pregnancies, with an overall correct sex prediction in 22/32 cases (68.7%). CONCLUSION: Fetal cells were not detected in a constant and reliable fashion in cervical mucus samples collected in the first trimester of pregnancy. The detection rate was poorly influenced by the use of different laboratory methods. This sampling technique cannot be regarded as a promising tool towards minimally invasive prenatal diagnosis.     

Comparison of two techniques for transcervical cell sampling performed in the same study population

OBJECTIVES: The aim of this study was to evaluate and compare the presence of fetal cells in transcervical cell (TCC) samples collected in the first trimester of pregnancy by two different procedures [mucus collection and intrauterine lavage (IUL)], performed consecutively in the same subjects scheduled for elective termination of pregnancy (TOP). METHODS: A total of 126 mucus/IUL sample pairs were retrieved from pregnant women immediately before TOP at a gestational age ranging from 7 to 12 weeks; at termination, samples of placental tissue were collected in all cases. All mucus samples were analysed by a polymerase chain reaction (PCR) assay and, in a subset of experiments involving 56 specimens, also by fluorescence in situ hybridization (FISH) procedure. IULs were divided in two aliquots, one for PCR analysis and one for the preparation of FISH slides. All placental tissue samples obtained at termination were analysed by FISH for fetal sexing. The PCR assay for fetal sex determination was performed by using, in a multiplex reaction, primers for SRY (Y chromosome sex-determining region, 738 bp) and HUMARA (human androgen receptor on the X chromosome, 280 bp) genes. The FISH analysis was carried out using direct-labelled commercial probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1, spectrum green) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1, spectrum orange) regions. RESULTS: In samples from known male pregnancies (n = 67), full concordance between IUL and mucus results could be found in 11 cases (16.4%); in 41 cases, Y chromosome material was detected by FISH (n = 2), by PCR (n = 5) or both (n = 34) in IUL samples, but not in the corresponding mucus samples. Y chromosome material was not documented in 10 mucus/IUL sample pairs. In 5 cases, the FISH (n = 2), the PCR (n = 1) or both (n = 2) failed to detect Y chromosome material in IULs, which was detected, however, by PCR in the corresponding mucus samples. Overall, correct sex prediction was achieved in 55/67 IULs (82%) and in 16/67 (23.9%) mucus samples from male pregnancies. In samples from known female pregnancies (n = 56), full concordance between results of IUL/mucus pairs and those on placental samples could be found in 53 cases (94.6%); in 3 cases, Y chromosome material was documented by PCR in mucus samples, but not in the corresponding IULs. Correct sex prediction was therefore achieved in 56/56 IULs (100%) and in 53/56 (94.6%) mucus samples from female pregnancies. CONCLUSION: This study provides evidence that, among TCC sampling techniques, IUL, but not mucus collection, can yield fetal cells in a constant and reliable fashion, which is a basic prerequisite for possible clinical usage. This suggestion had already emerged from some previous investigations but, owing to the study design, differences in study populations can no longer be used to explain the very different and sometimes-conflicting results reported in earlier studies.     

Examination of fetal cells and cell-free fetal DNA in maternal blood for fetal gender determination

BACKGROUND: To assess applicability of noninvasive methods for prenatal sex determination, both intact fetal cells and cell-free DNA from maternal blood were studied. METHODS: Maternal peripheral blood samples were obtained from 41 women carrying chromosomally normal fetuses and from 3 women with aneuploid fetuses (47,XX,+18; 47,XY,+18 and 47,XY,+21) at 9-22 weeks of gestation. DNA was extracted from the plasma fraction and analyzed by the nested polymerase chain reaction (PCR) using Y chromosome specific primers. After fetal cells were enriched by MACS, fluorescence in situ hybridization (FISH) with chromosome X and Y specific probes was performed to detect XY cells. RESULTS: Although Y-chromosome-specific DNA was detected by PCR analysis in all maternal plasma samples with male fetuses, 26% women bearing female fetuses also gave positive results. By FISH analysis, XY cells were detected in not only 58% of women bearing male fetuses, but also 13% of their counterpoints with female fetuses. CONCLUSIONS: Our findings suggested that consistent results for fetal gender using PCR or FISH cannot be obtained with intact fetal cells and cell-free DNA present in maternal blood and plasma at 9-22 weeks of gestation, despite their apparent abundant presence.     

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.     

Detection of fetal cells in intrauterine lavage samples collected in the first trimester of pregnancy.

OBJECTIVES: The aim of the present study was first to evaluate the presence of fetal cells in transcervical cell (TCC) samples collected by intrauterine lavage in the first trimester of pregnancy, and then to compare different methods for the detection of these cells. METHODS: TCC samples were collected by intrauterine lavage before termination of pregnancy (TOP) from 81 pregnant women between 7 and 12 weeks of gestation. Samples of placental tissue were collected from each patient at TOP, whereas maternal peripheral blood samples were obtained in 57 cases. DNA extracted from 81 lavage and the corresponding placental samples was amplified by a polymerase chain reaction (PCR) assay using primers for SRY and HUMARA genes. All 81 lavage samples were also analysed by fluorescent in situ hybridisation (FISH) using direct-labelled probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1) regions. In 57 cases, a quantitative fluorescent (QF) PCR assay, involving the use of two small tandem repeat (STR) markers (D21S11, D21S14.11) specific to chromosome 21 was employed to analyse DNA extracted from placental tissue, lavage and maternal blood samples. RESULTS: PCR analysis revealed that 40/81 placental samples were from male pregnancies. Correct sexing was achieved with the PCR technique in 30/40 (75%) lavage samples retrieved from pregnant women with male conceptuses and in all 41 (100%) samples collected from pregnancies with female fetuses. With the FISH analysis, nuclei bearing X and Y signals were observed in 32/40 cases (80%) from known male pregnancies, the rate of fetal cells ranging between 2% and 95%, whereas nuclei showing X and Y signals were not detected in any of the 41 lavage samples from known female pregnancies. Paternal peaks were present in 30/57 (52.6%) lavage samples tested by QF-PCR. CONCLUSION: The results suggest that fetal cells can be found, at a significant rate, in a very high proportion of intrauterine lavage samples. Therefore, this sampling technique can be regarded as a promising tool towards minimally invasive prenatal diagnosis. The FISH and PCR methods showed a similar efficiency in detecting fetal cells.     

Automated detection of rare fetal cells in maternal blood: eliminating the false-positive XY signals in XX pregnancies

OBJECTIVE: The purpose of this study was to develop a new method to help differentiate XX from XY signals in maternal blood from women carrying XY fetuses. STUDY DESIGN: We have developed a system to scan automatically for cells that bear X and Y fluorescence in situ hybridization signals. These XY target cells are identified by scans at low (x20) magnification, and all identified targets are revisited and verified at high (x100) magnification. The viewer software component of the system displays x20 images of all cells and intracellular fluorescence in situ hybridization signals that are present in each of the 4000 optical fields per slide, along with x100 images of automatically detected target cells. RESULTS: We initially examined 36,000 fields from 18 slides in 12 pregnancies (6 male and 6 female) using our system that is based on fluorescence in situ hybridization with a single probe for the X-chromosome and a single probe for the Y-chromosome and found XY nuclei in all samples, regardless of fetal gender. In the second phase of the study, a refinement of the approach that incorporated 2 independent probes for the Y-chromosome resulted in a false-positive rate for detection of XY nuclei in XX cases <0.00005%. CONCLUSION: Our data suggest that this system may allow for excellent "signal to noise" separation, which is required absolutely for fetal cell methods to differentiate aneuploid from normal pregnancies. Quantitation of fetal cells in the maternal circulation and standardization of processes that have been developed for their enrichment are crucial to moving fetal cell assessment from esoteric basic science to applied new technology.     

Endothelial precursor cells in the peripheral blood of pregnant women

OBJECTIVE: To determine whether primitive endothelial precursor cells are present in the peripheral blood of pregnant compared with nonpregnant subjects and whether these precursor cells are of fetal or maternal origin. METHODS: Peripheral blood mononuclear cells from 13 pregnant women in the second trimester and from ten nonpregnant women and men were cultured for 8-10 weeks under conditions that promoted endothelial cell development. Early outgrowth (1 week culture) and late outgrowth (4-6 weeks) colonies were observed, their endothelial nature was investigated, and fluorescence in situ hybridization was performed to determine the origin of the colonies from pregnant women's specimens. RESULTS: Peripheral blood mononuclear cell cultures from all pregnant women and all nonpregnant controls yielded early-outgrowth endothelial cells. Late-outgrowth endothelial cells were observed in 61.5% (eight of 13) of pregnant subjects, but in none of the ten nonpregnant controls (chi(2) test; P <.01). The adherent cells stained positively for von Willebrand factor and incorporated Dil-Ac-LDL, confirming their endothelial origin. Fluorescence in situ hybridization analysis showed only X chromosome-specific signals and no Y chromosome-specific signals in the cells from the late-outgrowth endothelial cells in all pregnant women carrying either a male (n = 5) or a female (n = 8) fetus. CONCLUSION: Primitive endothelial precursor cells are present in most pregnant women during the second trimester. These cells appear to be of maternal origin.     

Non-invasive first trimester fetal gender assignment in pregnancies at risk for X-linked recessive diseases

OBJECTIVES: Prenatal diagnosis in families affected by X-linked recessive disorders should ideally be limited to the subjects at increased risk, i.e. male fetuses, in order to avoid the risk of fetal loss due to the invasive procedure in healthy female fetuses. The aim of the study was to assess the fetal sex within the first trimester of gestation by two non-invasive approaches, using ultrasonography and a molecular analysis of fetal DNA extracted from whole maternal blood with specific markers, in order to avoid invasive sampling in female fetuses. METHODS: A total number of 18 fetuses at risk for an X-linked recessive disease were included in the present investigation. Maternal peripheral blood was analysed between 7 and 12 weeks of gestation by nested PCR for the detection of fetal DNA and the prediction of fetal gender. In addition, when the biparietal diameter (BPD) was between 21 and 23 mm, an ultrasonographic examination was carried out to assess the fetal gender. CVS was then performed in male fetuses only. RESULTS: Fetal gender was correctly assigned by ultrasonography between 21 and 23 mm of BPD in all the cases studied, whereas DNA extracted from whole maternal blood accurately predicted the gender in all the female cases (10), but failed in 4 out of 8 male fetuses, erroneously assigned as females. CONCLUSION: The present study shows that sonography is able to accurately predict the fetal gender within the first trimester of pregnancy, whereas the molecular analysis of DNA extracted from whole maternal blood is biased by false-Y-negative results in 50% of the cases.     

Detection of Y chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction

The present study was undertaken to evaluate a nested polymerase chain reaction (PCR) for detection of Y chromosome-specific fetal DNA in maternal plasma and urine of pregnant women during different gestational stages. DNA isolated from plasma and urine samples of 80 pregnant women (between 7 and 40 weeks' gestation) underwent amplification for Y chromosome-specific 198 bp DNA by nested PCR. The postpartum analysis of fetal gender showed that 55 women carried male and 25 female fetuses. Among the 55 women bearing male fetuses, Y chromosome-specific signals were detected in 53 (96%) plasma and 21 (38%) urine samples. Moreover, out of 25 women bearing female fetuses, 3 (12%) and 1 (4%) women had Y chromosome-specific signal in plasma and urine, respectively. Analysis of results with respect to gestational age revealed that there was no significant difference in the detection of Y chromosome-specific DNA between different trimesters in maternal plasma of women bearing male fetuses. These results showed that fetus-specific DNA was detected with high sensitivity (96%) and specificity (88%) in the maternal plasma by nested PCR, and therefore the method could be useful as a non-invasive procedure for fetal sex determination and prenatal diagnosis.     

A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double-density gradient system

OBJECTIVES: To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield. METHODS: Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer-Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X-Y probes. RESULTS: For single-density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H(2)O with 1.077 g/mL Percoll and 520 mOsm/kg H(2)O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double-Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus. CONCLUSION: The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods.     

Prenatal diagnosis from cystic hygroma fluid: the value of fluorescence in situ hybridization

OBJECTIVE: We sought to determine the optimal approach to the prenatal chromosome analysis of cystic hygroma fluid using traditional cytogenetic analysis and fluorescence in situ hybridization. STUDY DESIGN: A retrospective evaluation of our experience with traditional cytogenetic and fluorescence in situ hybridization analysis on cystic hygroma fluid was performed through a systematic review of the Genzyme Genetics database from January 1995 to July 2000. Information on gestational age, sample volume, clinical ultrasound findings (including fetal viability), cytogenetic results, fluorescence in situ hybridization results, and turn-around-time were queried. RESULTS: Eighty-three specimens were included in the investigation. The mean gestational age was 18.1 weeks (range, 13-27 weeks), and the mean sample volume was 20.7 mL (range, 0.1-101 mL). Of the 72 samples in which > 5 mL of cystic hygroma fluid was available, the success rate for cytogenetic analysis was 76% (55/72 samples). In 11 specimens of < or = 5 mL of cystic hygroma fluid, cytogenetic analysis was successful in only 1 case (9%). Fluorescence in situ hybridization was attempted on 23 samples, 18 of which were successful (78%), including 6 of 9 cases of cell culture failure (67%). Both traditional cytogenetic analysis and fluorescence in situ hybridization were performed in 21 instances when a sample of > 5 mL was available. A successful result was obtained by either cytogenetic testing or fluorescence in situ hybridization analysis or both in 19 of 21 of these cases (90%). Samples of > 5 mL from viable fetuses had a higher cytogenetic success rate (80%) and fluorescence in situ hybridization success rate (89%) than samples from fetuses with intrauterine death (38% and 50% cytogenetic and fluorescence in situ hybridization success rates, respectively.) The mean turn-around time was 8.2 days (range, 4-17 days). Results were available in < or = 12 days in 91% of cases. There was a 91% aneuploidy rate identified, with 45,X occurring in 86% of the samples. CONCLUSION: We conclude that the optimal approach for the prenatal diagnosis of chromosome abnormalities from cystic hygroma samples is to perform both traditional cytogenetic studies and interphase prenatal fluorescence in situ hybridization evaluation for the most common aneuploidies that involve chromosomes 13, 18, 21, X, and Y. With this combined approach, our data indicate that, in viable pregnancies with a fluid sample of >5 mL, a 90% diagnostic success rate can be achieved.     

Fetal nucleated red blood cells in peripheral blood of pregnant women: detection and determination of location on a slide using laser-scanning cytometry

OBJECTIVE: The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. METHODS: CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. RESULTS: In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. CONCLUSION: On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.     

The use of transferrin for enrichment of fetal cells from maternal blood

Iron loaded transferrin (holotransferrin) was used for enrichment of fetal cells from peripheral blood of pregnant women. Cord blood samples were used to evaluate enrichment efficacy of single and double MACS separations. Blood samples were obtained from 10 pregnant women prior to chorion villus sampling (CVS). Erythroblasts and other mononuclear cells were isolated by triple-density gradient centrifugation. Fetal cells were further enriched by positive magnetic sorting (VarioMACS) using biotinylated transferrin and streptavidin conjugated to magnetic microbeads. The isolated cells were analysed with dual-colour in situ hybridization (FISH) with X- and Y-chromosome specific probes. Male fetuses were correctly identified in three out of four (75%) pregnancies and female fetuses in six out of six (100%) pregnancies. By using transferrin instead of antibodies to the transferrin receptor to label and enrich fetal cells, we believe that unspecific binding attributed to immunological labelling can be minimized. The results indicate that transferrin may be an alternative to antibodies to transferrin receptor for separation of fetal cells from maternal blood.     

Trophoblast sampling by blind transcervical aspiration

Blind transcervical aspiration for trophoblast was carried out on 137 patients undergoing elective termination of early pregnancies. Trophoblast was obtained from 45 patients (33%) usually at the first attempt. Collection was not necessarily more successful between 8 and 11 weeks of gestation. In only 13 patients (9%) was trophoblast collected without contamination by maternal tissue, or blood. Perforation of the amniotic sac in one patient (1%), bleeding either observed, or detected histologically (34%) and introduction of infection (4%) constitutes a real threat to fetal survival. Maternal serum alpha-fetoprotein estimation appears useful and may forewarn likelihood of fetal damage. Although transcervical aspiration should encounter ready acceptability as an out-patient procedure, modifications in the technique are essential before clinical application for detection of gene, or chromosome anomalies can be considered.     

Trophoblast sampling by blind transcervical aspiration

Summary. Blind transcervical aspiration for trophoblast was carried out on 137 patients undergoing elective termination of early pregnancies. Trophoblast was obtained from 45 patients (33%) usually at the first attempt. Collection was not necessarily more successful between 8 and 11 weeks of gestation. In only 13 patients (9%) was trophoblast collected without contamination by maternal tissue, or blood. Perforation of the amniotic sac in one patient (1%), bleeding either observed, or detected histologically (34%) and introduction of infection (4%) constitutes a real threat to fetal survival. Maternal serum a-fetoprotein estimation appears useful and may forewarn likelihood of fetal damage. Although transcervical aspiration should encounter ready acceptability as an outpatient procedure, modifications in the technique are essential before clinical application for detection of gene, or chromosome anomalies can be considered.     

染色体13／21α卫得探针用于产前诊断21三体综合征

目的：探讨应用染色体13／21α卫星探荧光原位杂交（FISH）技术行产前论断21三体综合征的价值。方法：选择10例经产前细胞遗传学检查证实为孕正常胎儿孕妇的羊水细胞（对照组）、3例证实为21三体胎儿孕妇的羊水细胞（观察组）,用13／21α卫星探针对未经培养的羊水细胞间期核进行FISH杂交,结果：两组总杂交率分别为36．7％和38．6％,差异无显著性（P＞0．05）。对照组和观察组含4个杂交信号的核平均丰分比分别为36．5％和3．9％,含5个杂交信号的核平均百分比分别为4．0％和36．1％,差异有极显著性（P＜0．01）,含5个信号的百分比＜36．1％可作为21三体综合征的诊断标准。结论：13／21α卫星探针间期FISH用于未培养的羊不细胞可以快速,准确地在产前诊断21三体综合征。     

Integration of the transabdominal technique into an ongoing chorionic villus sampling program

Data are presented on 869 patients undergoing chorionic villus sampling procedures by one of two sampling techniques: 544 by a transcervical catheter aspiration method and 325 by a transabdominal two-needle aspiration method. The transcervical approach was the only procedure used in the first 330 cases, at which time the transabdominal technique was incorporated into our program. After an initial learning curve in the first 100 procedures the transcervical fetal loss rate stabilized at 2.7%, the number of patients requiring more than one catheter insertion decreased to 11%, and tissue weights greater than or equal to 10 mg were obtained in 88% of cases. The fetal loss rate for transabdominal chorionic villus sampling was 2.6%, indicating the addition of this new method did not significantly alter the fetal loss rate. Transabdominal chorionic villus sampling had an overall success rate of 99%, with only one insertion of the guide needle required for 98% of patients. Tissue weights of greater than or equal to 10 mg were obtained in 99% of cases. These results demonstrate that the transabdominal procedure can be rapidly and effectively incorporated by an operator already experienced with transcervical chorionic villus sampling. Since several contraindications exist for either chorionic villus sampling method, the availability of both techniques at a single center greatly enhances the ability to offer first-trimester fetal diagnosis to a majority of patients.     

Detection of fetal cells from transcervical mucus plug before first-trimester termination of pregnancy by cytokeratin-7 immunohistochemistry

AIM: The presence of fetal cells in the endocervical mucus of pregnant women was first reported in 1971. The uterine cavity is patent during the first trimester prior to fusion of amnion and chorion. Fetal cells from degenerating chorion frondosum are theoretically shed into the uterine cavity between seven and 13 weeks' gestation and are trapped in the transcervical mucus; they can be identified by immunohistochemistry. METHOD: Ninety-nine transcervical mucosal plugs from pregnant women of between 7 and 13 weeks before abortion were collected, fixed, embedded, sectioned and stained with monoclonal antibody of cytokeratin-7 (CK-7) by immunohistochemistry. RESULT: The identification of trophoblasts on each slide was defined according to positive staining and histologically chorionic villous similarity under microscopic examination, using the following five categories: none (1), less than five single positive-stained cells per-section (2), more than five single positive-stained cells per-section (3), clumps of positive-stained cells (4), and histological-like intact or fragmented chorionic villi (5). From amongst 71 samples that qualified for analysis, individual slides were evaluated and categorized into three groups, with the following results: 32 (45.1%) fell into group 1 (category 1) denoting a negative result, 10 (14.1%) fell into group 2 (category 2) indicating a possible positive result and 29 (40.8%) fell into group 3 (any combination of categories 3-5) representing a positive result. CONCLUSIONS: Fetal cells, identified by CK-7, can be found in more than 59.2% of the transcervical mucus in early pregnancy by use of a minimally invasive sampling method. Prenatal diagnosis of single-gene or chromosome disorders may be possible in the pregnant transcervical mucus by use of modern molecular methods and they deserve further study.     

Defining the role of fluorescence in situ hybridization on uncultured amniocytes for prenatal diagnosis of aneuploidies

OBJECTIVE: This study examines the role of fluorescence in situ hybridization on uncultured amniocytes for prenatal diagnosis in a population at high risk for aneuploidies.STUDY DESIGN: All patients undergoing amniocentesis for fetal structural abnormality on ultrasonographic examination (performed from 13 to 39 weeks), abnormal maternal serum aneuploidy screening results, or advanced maternal age with substantial parental anxiety were offered both fluorescence in situ hybridization on uncultured cells and conventional metaphase karyotyping on dividing cells.RESULTS: From 1992 to 1995, 315 patients were studied. Mean time to obtain results was 2.8 days for fluorescence in situ hybridization and 8.3 days for karyotype. Fluorescence in situ hybridization was informative in 254 samples (80.6%), and within this group 21 aneuploidies were correctly identified. Among informative specimens there was 100% sensitivity and specificity, with 100% positive and negative predictive values. Of the 315 samples, 61 (19.4%) were uninformative or unreportable. Of 25 total cases of karyotype-proved aneuploidy, 4 were reported as uninformative by fluorescence in situ hybridization, for a total detection rate of 84%. Overall, amniocenteses performed after 24 weeks were significantly more likely to be uninformative than those performed in the second trimester (45% vs 16%, p = 0.01), peaking at a 56% uninformative rate after 33 weeks. Logistic regression analysis showed an 8% increase in the uninformative rate per week of gestational age (odds ratio 1.08, 95% confidence interval 1.04 to 1.14).CONCLUSIONS: Fluorescence in situ hybridization on uncultured amniocytes is a rapid, clinically useful tool for prenatal diagnosis, with informative specimens being highly accurate. The combination of a structural fetal anomaly and an abnormal fluorescence in situ hybridization result should allow for definitive management decisions. The significant increase in uninformative specimens at later gestational ages limits its usefulness in the third trimester. (Am J Obstet Gynecol 1997;176:769-76.)