Trophoblasts circulating in maternal blood as candidates for prenatal genetic evaluation.
Trophoblasts have been detected in uterine venous blood, lung parenchyma and maternal blood in the first trimester. Their dilution within maternal leukocytes has been recently estimated to be 1:10(-6). The objectives of this study were to enrich peripheral maternal blood preparations for trophoblast cells, to isolate trophoblasts from the enriched preparation by highly specific markers and to assess fetal cell total number of chromosomal copies by fluorescence in-situ hybridization (FISH). Negative and positive selections for trophoblasts were performed. To assess the efficacy of the enrichment methods, a model mimicking the in-vivo conditions was established. Purified first trimester trophoblasts were prepared from first trimester placentas and were mixed with leukocytes from non-pregnant women in various concentrations. Magnetic beads coupled with antibodies to the common leukocyte antigen (CD45) or to antitrophoblast specific antigens (Trop1, Trop2 and GB25), were attached to peripheral maternal blood cells or to the prepared mixed cell populations. The expression of alpha human chorionic gonadotrophin (alpha HCG) or of human placental lactogen (HPL) by the remaining cells was examined by two means: (i) immunocytochemistry, using monoclonal antibodies against HPL and alpha HCG to stain fetal cells; (ii) reverse transcriptase polymerase chain reaction (RT-PCR), using specific primers for exons of the HPL or alpha HCG mRNAs. Results revealed that HPL- and alpha HCG-expressing cells could be identified in maternal blood only in very rare instances. On the other hand, expression of alpha HCG and HPL by only 100 purified first trimester trophoblasts artificially mixed with peripheral leukocytes at a ratio of 1:10(-5) could be identified by both immunocytochemistry and RT-PCR.(ABSTRACT TRUNCATED AT 250 WORDS)
Yagel
S
S
Department of Obstetrics and Gynaecology, Hadassah Mount Scopus, Jerusalem, Israel.
Shpan
P
P
Dushnik
M
M
Livni
N
N
Shimonovitz
S
S
D001483:Base Sequence; D002458:Cell Fractionation; D002875:Chromosomes; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D011247:Pregnancy / Q000097:blood*; D011296:Prenatal Diagnosis / Q000379:methods*; D014327:Trophoblasts / Q000502:physiology*