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


CONCLUSIONS
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.
Bussani
Cecilia
C
Department of Gynecology, Perinatology and Human Reproduction, University of Florence, Florence, Italy. cecilia.bussani@unifi.it
Scarselli
Benedetta
B
Cioni
Riccardo
R
Bucciantini
Sandra
S
Scarselli
Gianfranco
G
D002584:Cervix Uteri; D004247:DNA / Q000032:analysis*; D005260:Female; D005333:Fetus / Q000502:physiology*; D005453:Fluorescence; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008846:Micromanipulation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011296:Prenatal Diagnosis / Q000379:methods; D015203:Reproducibility of Results; D012732:Sex Determination Analysis / Q000379:methods; D007507:Therapeutic Irrigation
D004247:DNA