Use of the quantitative fluorescent-PCR assay in the study of fetal DNA from micromanipulated transcervical samples.

AIM: The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. METHODS: TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. RESULTS: Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). CONCLUSION: This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Detection of fetal DNA in the peritoneal cavity during pregnancy.

There is increasing evidence that fetal cells are commonly shed toward the cervix and in maternal circulation during pregnancy. In this study, a sample of peritoneal fluid was retrieved from a primigravida at 12 weeks' gestation undergoing urgent intervention for the torsion of an adnexal mass; the sample was then analysed by a polymerase chain reaction (PCR) assay using primers for X- and Y-chromosome specific sequences, and Y-derived sequences were identified. The course of pregnancy was then uneventful until term, when the patient delivered a male fetus, thus, supporting the hypothesis of a fetal origin for the Y-derived sequences detected in the peritoneal fluid. Further studies are required in order to confirm these findings and precisely define the origin of these sequences; however, this report seems to provide further evidence of the spreading of fetal cells during gestation and addresses relevant issues as to the possibility of collecting these cells by culdocentesis and intraperitoneal lavage for 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.

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.

Strategies for the isolation and detection of fetal cells in transcervical samples.

OBJECTIVE: The aim of the study was to evaluate the detection of fetal cells from transcervical samples, collected in early pregnancy, by means of different molecular techniques. The value of the isolation of trophoblasts using an inverted microscope, also referred to as micromanipulation, is discussed. METHODS: All the 89 specimens were obtained by intrauterine lavages before termination of pregnancy (TOP), between 7 and 12 weeks of gestation. Micromanipulation was carried out in a subgroup of 57 for the isolation of fetal material. Fetal sexing was achieved by FISH (fluorescent in situ hybridisation) using fluorescently labelled probes for X and Y chromosomes and by polymerase chain reaction (PCR). Male samples were also investigated for aneuploidy of the chromosome 21. Quantitative fluorescent (QF)-PCR using two short tandem repeat (STR) markers for chromosome 21 was carried out in 26 micromanipulated samples. RESULTS: FISH analysis revealed that 45/89 placental samples derived from pregnancies with male fetuses. Correct sexing of the lavage samples from male pregnancies was achieved in 41/45 (91%) using dual-FISH technique, and in 43/45 (95.5%) with PCR. All the samples derived from male pregnancies tested for chromosome 21 were normal. From 57 samples subjected to micromanipulation, 51 (89.5%) showed discernible chorionic villous filaments or cell clumps of possible trophoblastic origin. One case of tetraploidy and two cases of monosomy were recorded. The rate of fetal cells, in the non-micromanipulated samples, was between 4% and 97% (mean 54.3%). In micromanipulated specimens, maternal contaminant cells were absent or extremely rare (1-2%). The efficiency of the QF-PCR analysis in detecting paternal peaks in all lavage samples was only 61.5%. CONCLUSION: The present study confirms the presence of fetal cells in a very high proportion of both whole and micromanipulated intrauterine lavage samples. The isolation of trophoblastic elements can be achieved in most cases by micromanipulation. FISH and PCR techniques allowed the analysis of the most common fetal aneuploidies, confirming the power of this minimally invasive method.

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.

HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy.

Different mechanisms mediated by the expression of the HLA-class Ib HLA-G products are suggested to account for the induction of immune tolerance against the paternal antigens of the fetus during pregnancy. Soluble HLA-G antigens, mainly produced by cytotrophoblast cells at the materno-fetal interface and circulating in the body fluids, show a capacity analogous to that of membrane-boundstructures to inhibit NK cells. In the present report we have investigated, using specific ELISA, the presence of sHLA-G molecules in culture supernatants of early embryos obtained by in vitro fertilization (IVF) before transfer. The data obtained from the analysis of 285 supernatants corresponding to 101 IVF procedures (43 IVF, 58 intracytoplasmic sperm injection) identify two groups of patients on the basis of sHLA-G antigen presence. No differences in clinical parameters were observed between the groups, but positive embryo implantations occurred only in women showing sHLA-G molecules in culture supernatants (Fisher's exact p value 2.56 x 10(-3)). The results obtained indicate that expression of HLA-G products in embryo cells is a mandatory, but not sufficient, prerequisite for the development of pregnancy.