Oligonucleotide array analysis of
miscarriages.
Robberecht C1, Mattheeuws E1, Fryns JP1, Vermeesch JR1
1
Center for Human Genetics, K.U.Leuven-UZ Gasthuisberg, Leuven, Belgium
Introduction & methods
Spontaneous abortions are common, with 10% to 15% of all clinically recognized pregnancies ending in early pregnancy loss. Cytogenetic analysis has shown that about
50-60% of first trimester miscarriages are caused by fetal chromosome abnormalities, most of which consist of numerical abnormalities (86%), including trisomies,
monosomies and polyploidies. Routine analysis of miscarriage samples has been performed by karyotyping of metaphase spreads after tissue culture. However, due to
failure of culture growth, suboptimal chromosome preparations, or possible maternal cell overgrowth often no result or an erroneous result is obtained.
We therefore implemented array comparative genomic hybridisation (array CGH) using an Oxford Gene Technology (OGT) 15K oligonucleotide array with a
detection limit around 250kb, combined with interphase fluorescent in situ hybridisation (FISH) using X & Y probes. This technique avoids these cell culture problems and
reduces the turn around time. It can also improve the pick up rate because it allows for the detection of submicroscopic aberrations through the use of a higher resolution
platform and for a better detection of mosaicism. We evaluated this technique
p
q on 137 miscarriage
g samples.
p
Results:
•Aberrations found in 40% of samples.
•Diverse range of trisomies (table) with 2 double
trisomies. Some trisomies are frequent findings
(chrom. 15, 16, 21) while others are rarely if ever
detected.
•Rare monosomy 4. Most monosomies are selected
against during early pregnancy,
pregnancy many even before
implantation.
•Detection of segmental aberrations, though none were
submicroscopic (figure 2).
•Skewed male (1/4) to female (3/4) ratio in the normal
samples.
Polyploidy detected by array CGH
92,XXYY(15%)/46,XY(15%)/46,XX(70%) vs. 47,XXY ref.
69,XXY vs. 47,XXY ref.
XY
Fig 1A
Fig 1B
X
Y
Figure 1A & B: Array CGH profiles using DNA from a triploidy (A) and a tetraploidy (B) versus DNA from an 47,XXY cell line. For each panel, the x-axis represents the
oligonucleotide probes ordered from the short-arm telomere to the long-arm telomere and chromosomes are ordered from chromosomes 1 to 22, X and Y. The y-axis shows log2
transformed intensity ratios at each locus (patient Cy5/control Cy3). 69,XXY triploidy samples always show an equal drop of the X and Y ratios, while 92,XXYY samples are
y indicated byy different deletion ratios depending
p
g on the p
percentage
g of maternal cells.
usually
Segmental aberrations detected by array CGH
47,XX,+19;del(2)(q31.1q33.1) vs. 47,XXY ref.
Fig 2A
47,XX,+i(5p)(20%)/46,XX(80%) vs. 47,XXY ref.
Fig 2B
Figure 2A & B: Array CGH profiles using DNA from a chromosome 2 segmental deletion (A) and an isochromosome 5p (B) versus DNA from an 47,XXY cell line. For each panel,
the x-axis represents the oligonucleotide probes ordered from the short-arm telomere to the long-arm telomere . The y-axis shows log2 transformed intensity ratios at each locus
(patient Cy5/control Cy3).
Discussion:
•Array CGH overcomes many limitations of traditional karyotyping. No cell culture is needed, so there is no chance of culture failure, culture contamination, maternal cell
overgrowth or selection against abnormal cells in mosaic cases. The increased resolution of this method also allows for the detection of smaller aberrations.
•We demonstrate that detection of triploidy (69,XXY, not XXX) and tetraploidy (all 92,XXYY) is possible when a 47,XXY reference is used, but a second technique, such as
interphase FISH or flow cytometry, is often required to confirm the result or to distinguish it from a male sample with maternal cell contamination.
• 15K OGT oligonucleotide arrays are easy to use. They are available on an 8x15K slide and can be analyzed in 2-3days, creating a fast routine work-flow.