Analysis of human invasive cytotrophoblasts using multicolor fluorescence in situ hybridization

Abstract

Multicolor fluorescence in situ hybridization, or FISH, is a widely used method to assess fixed tissues or isolated cells for numerical and structural chromosome aberrations. Unlike other screening procedures which provide average chromosome numbers for heterogeneous samples, FISH is a sensitive cell-by-cell method to analyze the distribution of abnormal cells in complex tissues. Here, we applied FISH to characterize chromosomal composition of a rare, but very important class of human cells that stabilize the fetal-maternal interface connecting the placenta to the uterine wall during early pregnancy, called invasive cytotrophoblasts (iCTBs). Combining differently-labeled, chromosome-specific DNA probes, we were able to unambiguously determine the number of up to six different autosomes and gonosomes in individual cell nuclei from iCTBs selected on the basis of their invasive behavior. In this manuscript, we describe a method for generation of iCTBs from placental villi, and provide the complete workflow of our FISH experiments including a detailed description of reagents and a trouble-shooting guide. We also include an in-depth discussion of the various types and sources of DNA probes which have evolved considerably in the last two decades. Thus, this communication represents both a complete guide as well as a valuable resource, intended to allow an average laboratory to reproduce the experiments and minimize the amount of specialized, and often costly, equipment.

Keywords: 4,6-diamino-2-phenylindole; Aneuploidy; BAC; CPM; CTB; Cytotrophoblast; DAPI; DNA probes; FISH; HLA-G; In situ hybridization; LB; Luria Bertone; Placenta; TB; bacterial artificial chromosome; confined placental mosaicism; cytotrophoblast; fluorescence in situ hybridization; human leukocyte antigen G; iCTB; invasive cytotrophoblast; trophoblast.