Analysis of human invasive cytotrophoblasts demonstrates mosaic aneuploidy

Abstract

A total of 24 chromosome-specific fluorescence in situ hybridization probes for interphase nucleus analysis were developed to determine the chromosomal content of individual human invasive cytotrophoblasts derived from in vitro cultured assays. At least 75% of invasive cytotrophoblasts were hyperdiploid and the total number of chromosomes ranged from 47 to 61. The results also demonstrated that these hyperdiploid invasive cytotrophoblasts showed significant heterogeneity. The most copy number gains were observed for chromosomes 13, 14, 15, 19, 21, and 22 with average copy number greater than 2.3. A parallel study using primary invasive cytotrophoblasts also showed a similar trend of copy number changes. Conclusively, 24-chromosome analysis of human non-proliferating cytotrophoblasts (interphase nuclei) was achieved. Hyperdiploidy and chromosomal heterogeneity without endoduplication in invasive cytotrophoblasts may suggest a selective advantage for invasion and short lifespan during normal placental development.

Fig 1. First trimester placental villi grown on Matrigel to obtain invasive cytotrophoblasts (iCTBs) in vitro.

(A, B) Representative images of iCTBs outgrowth from a 6.4wk placental villi culture explants for 3 days. (C) Immunostaining of first trimester placenta villi culture explant showed most iCTBs are positive for Cytokeratin-7 (CK-7). (D) CTBs on the tip of the villi differentiated into iCTBs were harvested and fixed on glass slides showing positive for HLA-G.

Fig 2. The mean copy number of each chromosome for normal male lymphocytes and invasive cytotrophoblasts derived from in-vitro Matrigel culture.

Invasive CTBs with significant gain of extra copies of chromosomes 2, 3, 5, 10, 13, 14, 15, 19, 21 and 22. (*) (P < 0.05). Samples contained a total of 100 lymphocytes and 600 invasive CTBs from two female and three male placentas.

Fig 3. Analysis of 24 chromosomes on one invasive cytotrophoblast using fluorescence in situ hybridization.

(A-D) FISH probe sets I to IV were hybridized on the same invasive CTB by sequentially repeated hybridizations. (E-F) The corresponding classified image from Spectral Imaging system (pseudo-colors) revealed the following hyperdiploid karyotype for this CTB: nuc ish 56,XY,+1,+3,+5,+5,+5,+10,+10,+15,+19,+19.

Fig 4. Total number of chromosomes for four different invasive cytotrophoblasts.

At least 75% of the male cells and 54% of the female cells were hyperdiploid (Total number of chromosomes > 46). Male iCTBs-1: 98 cells; Male iCTBs-2: 91 cells; Female iCTBs-3: 57 cells; Male iCTBs-4: 83 cells). Y-axes represent the Fraction of cells.

Fig 5. Hyperploid rate of each chromosome on invasive cytotrophoblasts.

The most common chromosomal gains affected chromosomes 13, 14, 15, 19, 21, and 22. Samples contain 600 invasive CTBs from two female and three male placentas.

Fig 6. Fluorescence in situ hybridization analysis of second trimester placental tissue sections.

Thirteen different chromosome probes were hybridized on tissue sections showed that hyperdiploidy was found more often in cells of the uterine wall (BP, invasive cells in basal plate) than in those of the floating villi (ST: syncytiotrophoblasts; VC: mesenchyme in villus core). Significant differences between the means were found on chromosomes 19, 22, 15, 14, 21, 13, and 8. (*) (P < 0.025). Each chromosome was tested on 2–6 different second trimester placentas.

Fig 7. The hyperdiploidy rate on different chromosomes for invasive CTBs in vivo and in vitro.

The hyperdiploidy rate of different chromosomes in invasive CTBs correlated with in vivo tissues (2–6 samples) and in vitro studies (5 samples). The most common chromosomal gains were found in chromosomes 15, 19, and 22. Significant differences between the means were found on chromosomes 6 and 9. (*) (P < 0.05).