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ORIGINAL ARTICLE
Implementation of a High-Resolution Single-Nucleotide
Polymorphism Array in Analyzing the Products of Conception
HuiMin Zhang,
1,2
WeiQiang Liu,
2
Min Chen,
2
ZhiHua Li,
2
XiaoFang Sun,
2
and ChenHong Wang
1,3
Aims: To demonstrate the value of a whole-genome high-resolution single-nucleotide polymorphism (SNP)
array for the elucidation of genetic causes underlying pregnancy loss. Methods: The SNP array combined with
SNPs and oligonucleotide probes was used to examine 60 samples of products of conception, including
chorionic villi, fetal parts, and fetal blood. Results: The SNP array yielded a 38.3% (23/60) abnormality rate. In
addition to the most common aneuploidy, it detected 16.7% additional aberrations involving copy number
variation, triploidy, loss of heterozygosity or low-level mosaicism. Conclusion: This whole-genome high-
resolution SNP array not only provides copy number information but also identifies the heterozygosity status,
which facilitates the discovery of the novel genetic alterations associated with pregnancy failure and improves
the management of subsequent pregnancies.
Introduction
Spontaneous abortion and stillbirth are common
occurrences during pregnancy. Approximately 15–20%
of all clinically recognized pregnancies end in miscarriage,
with most occurring during the first trimester. If biochemical
pregnancy is taken into account, the pregnancy failure rate
increases 4–5 times (Simpson and Juneau, 2012; Stock,
2012). It is well understood that pregnancy is a complex
process involving the fetus, mother, and placenta and that
genetic factors appear to play the most important role.
Cytogenetic analysis of the products of conception (POC)
from pregnancy failure has indicated that severe genomic
imbalances caused by embryonic chromosomal abnormali-
ties account for *50% of first trimester miscarriages; of
these abnormalities, 86% are numerical abnormalities, 6%
are structural abnormalities, and 8% are other chromosomal
abnormalities, including mosaicism (Goddijn and Leschot,
2000). Understanding the causes of fetal loss may be in-
valuable because it may eliminate further testing and provide
a better estimate of the recurrence risk for couples.
Karyotyping is considered to be the ‘‘gold standard’’ for
detecting microscopic chromosomal aberrations. However, it
is only able to detect chromosomal changes of *5–10 Mb; in
addition, many factors, including cell culture failure, selec-
tive growth of abnormal or maternal cells, poor chromosome
morphology, and subjective errors of technicians, could in-
fluence the success rate or accuracy of conventional kar-
yotyping (Menasha et al., 2005). Furthermore, a previous
study has shown that *20% of samples cannot be karyotyped
because of culture failure (Shearer et al., 2011).
Recently, there have been many reports about the clinical
applications of the following methods for testing POC
specimens: fluorescence in situ hybridization (FISH), quan-
titative fluorescence polymerase chain reaction, and multi-
plex ligation-dependent probe amplification (Donaghue
et al., 2010; Jobanputra et al., 2011; Kim et al., 2015).
However, because these methods are limited to specific loci
on particular chromosomes, they cannot find aberrations at
the whole-genome level. With the increasing resolution of
molecular methods, the possibility has been raised that mis-
carriages could be due to submicroscopic chromosomal ab-
errations (Do
´ria et al., 2009; Rajcan-Separovic et al., 2010a,
2010b). In addition, the association between copy number
variations (CNVs) and pregnancy failure is a concern.
The chromosomal microarray analysis (CMA) approach
uses extracted DNA, which represents the actual condition of
the tissue, overcomes many of the limitations of conventional
cytogenetic analysis on POC specimens, enhances the suc-
cess rate of the test, and improves the turnaround time and the
detection of submicroscopic chromosomal aberrations. It can
provide comprehensive and detailed results at high resolution
at the whole-genome level (Benkhalifa et al., 2005). Some
studies have indicated that CMA can detect 5–13% of the
additional chromosome abnormalities that are undetectable
by karyotyping (Dhillon et al., 2014). One study showed that
CMA could detect abnormalities in 29–50% of samples from
failed tissue cultures, and 6% of those samples exhibited
1
Graduate School, Southern Medical University, Guangzhou, P.R. China.
2
Key Laboratory for Reproduction and Genetics of Guangdong Higher Education Institutes, Key Laboratory for Major Obstetric Diseases
of Guangdong Province, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, P.R. China.
3
Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, P.R. China.
GENETIC TESTING AND MOLECULAR BIOMARKERS
Volume 20, Number 7, 2016
ªMary Ann Liebert, Inc.
Pp. 1–7
DOI: 10.1089/gtmb.2016.0035
1
submicroscopic aberrations (Rajcan-Separovic et al., 2010b).
Therefore, CMA has been recommended as a preferred
method for the analysis of POC specimens ( Menten et al.,
2009; Robberecht et al., 2009; Shaffer et al., 2012).
The CMA approach includes array-based comparative
genomic hybridizations (CGH) and single-nucleotide poly-
morphism (SNP)-based arrays. In contrast, to be validated in
the postnatal and prenatal areas, most studies applied targeted
low-resolution array CGH to the POC specimens. We applied
the whole-genome high-resolution SNP array combined with
SNP and oligonucleotide probes, which not only provide
copy number information but also identify triploidy, loss of
heterozygosity (LOH), uniparental disomy (UPD), and low-
level mosaicism.
This study was designed to identify possible miscarriage-
associated aberrations using an SNP array on 60 POC
specimens and elucidate more of the genetic mechanisms
underlying pregnancy failure to help couples in subsequent
pregnancies.
Materials and Methods
Sample information
The SNP array analysis was performed on samples from a
total of 60 cases diagnosed as abortions or stillbirths; the
samples included chorionic villi, fetal parts, and fetal blood.
Before the initial study, the hospital Medical Ethics Com-
mittee checked and approved the research. Pretest genetic
counseling about the benefits and limitations of CMA was
carried out with the couples, and informed consent was ob-
tained. According to the procedure, the villi and tissue sam-
ples were washed thrice with a phosphate-buffered solution
and placed on the stage of an inverted dissecting microscope
for the careful removal of decidua and blood clots to reduce
the risk of maternal cell contamination. Samples were col-
lected for genomic DNA extraction with the Qiagen DNeasy
Tissue Kit according to the manufacturer’s instructions
(Qiagen, Hilden, Germany).
SNP-based microarray analysis and data interpretation
The genome-wide human CytoScan 750K array (Affy-
metrix, Santa Clara, CA), containing >750,000 markers
combined with 550,000 unique nonpolymorphic oligonucle-
otide probes and 200,000 SNP probes, was used to investi-
gate the CNVs and SNPs. The microarrays were prepared
according to the manufacturer’s protocol (Affymetrix), in-
cluding digestion, ligation, amplification, fragmentation, la-
beling, hybridization, and scans. The raw data were initially
analyzed and viewed using the Affymetrix Chromosome
Analysis Suite (ChAS 2.2; Affymetrix). The reporting
threshold of the copy number was set at 300 kb. ChAS was set
to display LOH >3 Mb in size, and the cutoff for a single LOH
of >10 Mb on a single chromosome indicated UPD.
We evaluated the CNV with publicly available online
databases, which included the Database of Genomic Variants
(DGV), the Database of Chromosome Imbalance and Phe-
notype in Humans using Ensembl Resources (DECIPHER),
the International Standards for Cytogenomic Arrays (ISCA)
Consortium, Online Mendelian Inheritance in Man (OMIM)
genes, and RefSeq genes. According to the guidelines re-
commended by the American College of Medical Genetics
(ACMG), we interpreted the CNV as pathogenic, benign, or
variants of uncertain clinical significance (VOUS).
Statistical analysis
SPSS version 13 for Windows (SPSS, Inc., Chicago, IL)
was utilized to perform chi-square tests; a p-value <0.05 was
considered to be statistically significant.
Results
Successful results from the whole-genome high-resolution
SNP array were obtained from chorionic villi, fetal parts, and
fetal blood for all 60 POC specimens. The mean gestational
age was 17.2 weeks, the mean age of the women was 29.9
years, and the mean number of pregnancies was 2.2. The
results yielded a 38.3% (23/60) abnormality frequency. The
detailed results are shown in Table 1.
Of the abnormalities, the most common were aneuploidy,
including trisomy 21, 18, 13, 16, 15, and 2, double trisomy,
Turner syndrome (XO), and Klinefelter syndrome (XXY)
(Fig. 1A–C). In addition, the SNP array identified six CNVs
ranging between 241 kb and 23 Mb in size, including two
cases of loss, three cases of gain, and one case of gain
combined with loss, which corresponded to an unbalanced
chromosomal rearrangement, a region rich with imprinted
genes on 11p15.5 and the genes ZIC2,ENPP1,GJA1, and
FBN1 (Figs. 1D–F and 2). In addition to two cases of VOUS,
we identified four cases of CNVs as pathologic. The complete
list of CNVs, including their sizes, locations, and gene con-
tents, is shown in Table 2.
In contrast to array CGH, the SNP array identified addi-
tional abnormalities, including two cases of triploidy from
the abortion samples, one case of LOH from the sample with
mild fetal lateral ventriculomegaly, and one case of mosaic
trisomy 13 (*20%) from the sample of a twin pregnancy. In
Table 1. Characteristics of the SNP Array
Results from the 60 POC Specimens
Result No. of cases Percentage
No abnormality 37 61.6
Aneuploidy 13 21.7
Trisomy 21 2
Trisomy 18 1
Trisomy 13 1
Trisomy 16 2
Trisomy 15 1
Trisomy 2 1
Double trisomy 7, 14 1
Turner syndrome (XO) 3
Klinefelter syndrome (XXY) 1
Triploidy (XXX/XXY) 2 3.3
CNV 6 10
Pathogenic 4
VOUS 2
Mosaicism 1 1.7
LOH 1 1.7
Total 60 100
CNV, copy number variation; LOH, loss of heterozygosity; POC,
products of conception; SNP, single-nucleotide polymorphism;
VOUS, variants of uncertain clinical significance.
2 ZHANG ET AL.
all, the SNP array resulted in a 16.7% increase in the fre-
quency of abnormalities detected (Fig. 3).
There were 22 first-trimester cases and 38 second- or third-
trimester cases among the POC samples. The SNP array re-
sults showed that the frequencies of aneuploidy, CNVs, and
mosaicisms in the first trimester were 54.5% (12/22), 45.5%
(10/22), 4.5% (1/22), and 4.5% (1/22), respectively, and in
the second or third trimester they were 26.3% (10/38), 13.2%
(5/38), 13.2% (5/38), and 0% (0/38), respectively, including
one case of LOH. Statistical analysis showed that the fre-
quency of aneuploidy plus triploidy between the first and the
second- or third-trimester groups was significantly different
(P<0.05) and that there was no significant difference be-
tween the frequencies of abnormalities and CNVs (P>0.05).
The results are shown in Table 3.
Discussion
In our study, the SNP array was performed successfully on
all 60 POC specimens and was not affected by the type or
quality of the specimens. It yielded a 38.3% abnormality
frequency;in addition to the most common aneuploidy, 16.7%
of the aberrations involved CNVs, triploidy, LOH, and low-
level mosaicism. Considering that most of the samples came
from the late stage of pregnancy, the abnormality rate was
relatively low. Previous studies have shown that CMA can find
more submicroscopic aberrations related to pregnancy failure
and improve the detection rate in POC specimens. Further-
more, more CNVs of smaller sizes can be found (Perry et al.,
2008). Viaggi et al. (2013) detected additional aberrations not
identified by karyotyping in 15% of the POC specimens.
It should be noted that CMA results with POC specimens
from different studies vary; the additional frequencies of
abnormalities detected in other studies were 9.8% (Schaeffer
et al., 2004), 6–12% (Rajcan-Separovic et al., 2010b), and 5–
13% (Dhillon et al., 2014). However, in our study, it was
slightly higher than those reported in previous studies, which
could be related to differences in the types of microarray
platforms and the probe densities. In contrast to the targeted
low-resolution array CGH used in previous studies, our study
applied a whole-genome high-resolution SNP array com-
bined with oligonucleotide and SNP probes that not only
provided copy number information but also identified trip-
loidy, LOH, and UPD; therefore, the identification of more
abnormalities would be expected.
We identified six cases of CNVs involving chromosomal
rearrangements, a richly imprinted gene region on 11p15.5,
and the ZIC2,ENPP1,GJA1, and FBN1 genes. As is well
known, the proper development of the placenta involves
numerous genes (Hemberger, 2007). Studies in both human
FIG. 1. SNP array analysis of POC samples 1 and 2. (A) ChAS revealed a copy number gain occurring in the whole
chromosome 16 (blue arrow). (B) The three groups of orange lines, corresponding to weighted log
2
ratio, copy number
state, and smooth signal, represent the copy number of three for chromosome 16. Four allele peak signals clearly show the
presence of a copy number gain. (C) The whole genome view shows increased blue signals and four allele peaks in
chromosome 16 of sample 1 (blue arrows). (D) ChAS shows a copy number loss in the distal region of 1p and a copy
number gain in the distal region of 15q, indicating a chromosome rearrangement. (E, F) The allele peak signals and the
whole genome view further confirmed the copy number variants in the above region of sample 2. POC, products of
conception; SNP, single-nucleotide polymorphism. Color images available online at www.liebertpub.com/gtmb
IMPLEMENTATION OF SNP ARRAY IN POC 3
and model organisms have revealed that genes within the
CNV regions are expressed at more variable levels than
normal and CNVs not only affect the expression of genes that
they encompass because of copy number changes but also can
influence the transcriptome (Henrichsen et al., 2009). Studies
have shown that the disruption of genes, including genes
coding for growth factors, transcription factors, extracellular
matrix proteins, and signal proteins, results in embryonic
lethality. Numerous genes are up- or downregulated during
differentiation (Cross et al., 2003; Gheorghe et al., 2010).
Therefore, microarray analyses have provided insights into
the genetic mechanisms underlying pregnancy failure that
involve morphogenesis, organogenesis, angiogenesis, cellu-
lar transport, growth, and maintenance.
The ZIC2 gene is one of the five members of the zinc finger
family. The encoded zinc finger transcription factor plays an
important role in the process of neurodevelopment and par-
ticipates in the formation of the neural tube and crest. In the
human population, heterozygous mutation of the ZIC2 gene
or heterozygous deletion of the 13q32 region can lead to
holoprosencephaly.
The region from 6q22.1 to 23.2 is gene rich and contains
the ENPP1 and GJA1 genes associated with heart develop-
ment. The ENPP1 gene encodes a type II transmembrane
glycoprotein. Mutations in this gene cause extensive arterial
calcification resulting in cardiovascular issues, including
heart failure, hydropsy, hypertension, and extravascular
conditions. In addition, the GJA1 gene encodes connexin-43
(Cx43), one of the major proteins of gap junctions in the
synchronized contraction of the heart and in embryonic de-
velopment. Mutations of this gene lead to functional or de-
velopmental abnormalities of the heart.
The FBN1 gene encodes the fibrillin protein, a major
component of microfibrils of the extracellular matrix. Mu-
tations in the FBN1 gene are associated with the development
of Marfan syndrome. The possible underlying mechanism is
interference with the polymerization of fibrillin and the ag-
gregation of microfibrils.
The 11p15.5 region is rich with imprinted genes and plays
an important role in the maintenance of the fetus and pla-
centa; as a maternal–fetal exchange organ, the placenta plays
an important role in the growth and development of the fetus.
If aberrant methylation or the duplication of imprinted genes
occurs in this region, it will result in fetal demise.
Without access to parental samples, the CNVs in our study
could not be assessed as de novo or inherited. However, some
studies show that confirmed de novo CNVs in POC cases are
rare, small, and do not contain obvious candidate genes
FIG. 2. Microdeletions identified in POC samples 3 and 4. (A) ChAS indicated a copy number loss occurring in the
13q32.2q34 region (red arrow). (B) The red rectangle and reduced signals demonstrate that a copy number loss occurred in
this region. In addition, the three groups of blue signal lines represent the copy number, and the two allele peak signals in this
region further confirm a copy number loss in 13q32.2q34. (C) Chromosomal microarray analysis revealed the precise
breakpoint locations, the genes involved, and the deletion size of the 13q32.2q34 region in the sample 3. (D, E, F) Micro-
deletions in the 6q22.1q23.2 region were observed for sample 4. Color images available online at www.liebertpub.com/gtmb
4 ZHANG ET AL.
related to miscarriage in contrast to the CNVs found in
postnatal cases with developmental abnormalities, in which
the majority of the pathogenic CNVs are larger than 1 Mb.
However, the vast majority of CNVs of pregnancy failures
were confirmed as familial in origin, which could be associ-
ated with a detrimental effect on the embryo or placental de-
velopment, but not on the carrier parent, owing to imprinting,
recessive mutations on the remaining allele, and variable ex-
pressivity (Rajcan-Separovic et al., 2010a, 2010b).
Considering that the number of POC specimens that have
undergone cytogenetic analysis, especially those with pa-
rental verification, is limited, the CMA analysis of miscar-
riages faces several challenges. One of these challenges is the
lack of CNV data for successful pregnancies as controls.
Even in the DGV database, the fertility of the ‘‘normal’’
population is unknown. Therefore, a CNV database con-
taining fertility and pregnancy information is imperative.
Some CNVs that appear to be ‘‘benign’’ may produce dif-
ferent effects for internal changes in genes, changes in splice
sites, and the generation of different variants and new gene
products (Perry et al., 2008), making it difficult to determine
the properties of the CNVs. However, the clinical evaluation
of CNVs will become easier as knowledge of the human
genome increases (Bug et al., 2014).
In our study, of the 15 POC cases with numerical chro-
mosome abnormalities, 10 cases occurred in the first tri-
mester. Among the six POC cases with CNVs, five cases
occurred in the second or third trimester, and CNVs smaller
than 500 kb were identified in three samples. There was a
significant difference in the frequency of chromosome
number aberrations between the first trimester and the second
or third trimester. Therefore, it can be inferred that abnor-
malities involving whole chromosomes or large-sized CNVs
have a greater influence on genomes and inclined to preg-
nancy failure in the early stage and smaller CNVs have less
influence on genomes and are more likely to result in preg-
nancy loss in the later stage.
Compared with conventional array CGH, whole-genome
high-resolution SNP array can detect additional triploidy,
LOH, and UPD. As one of the major causes of pregnancy
failure, triploidy accounts for *10–20% of POC (Stephenson
et al., 2002). Two cases of triploidy were identified in this
study.
In addition, SNP-based analysis revealed a large portion of
homozygosity distributed in multiple regions of several
chromosomes from a stillbirth sample that had presented with
mild fetal lateral ventriculomegaly detected by ultrasound.
Based on the fact that the LOH region accounted for 8% of
the whole genome, it can be speculated that there was a
consanguineous marriage within three degrees of consan-
guinity (Kearney et al., 2011). Thus, it can be inferred that
LOH is likely to cause fetal demise through the homozygous
expression of autosomal recessive alleles (Engel, 1980).
However, it requires further verification.
In addition, a UPD case can be predicted if the homozy-
gosity is restricted to large blocks on a single chromosome,
which may lead to disorders by disrupting the balance of
imprinted genes. Systematic studies reported similar low
frequencies of LOH or UPD in POCs, which were estimated
to occur in <1% of pregnancy losses, and therefore, LOH or
UPD is possibly underestimated as a pathogenic factor in
fetal loss (Shaffer et al., 1998; Fritz et al., 2001; Levy et al.,
Table 2. CNVs Identified in the POC Specimens
Number
Condition of pregnancy
Clinical manifestation
CNV condition
SexAge Gestational age Gravidity Parity Locus
Type
of CNV
Size
(kb)
Genes
involved Property
1 26 16 1 0 Holoprosencephaly 13q32.2q34 Loss 16,000 ZIC2 Pathogenic XY
2 28 16 4 2 Hydroderma and nuchal
lymphatic hygroma
6q22.1q23.2 Loss 18,000 ENPP1 Pathogenic XX
GJA1
3 32 20 3 0 Stillbirth 15q21.1 Gain 295 FBN1 Pathogenic XY
4 37 12 5 0 Missed abortion 1p36.33p36.23 Loss 8000 Rearrangement Pathogenic XY
15q25.1q26.3 Gain 23,000
5 24 20 4 0 Stillbirth 11p15.5 Gain 363 Imprinted gene VOUS XX
241
6 36 14 2 0 Missed abortion 2p25.3 Gain 487 — VOUS XX
IMPLEMENTATION OF SNP ARRAY IN POC 5
2014). However, the real occurrence in POCs is also un-
known because of the detection limits of the available
methods. In some cases, the pathogenic relevance of LOH or
UPD is difficult to evaluate given the current state of
knowledge about the human genome. Therefore, it will be
important to establish an LOH or UPD database related to
pregnancy loss (Hemberger, 2007).
In our study, a low-level (*20%) mosaic case of trisomy
13 was identified in a specimen from a twin pregnancy fail-
ure. In contrast to karyotyping, the CMA method analyses the
extracted DNA, which represents the original state of the
tissue; therefore, more low-level mosaicism can be detected.
CMA cannot detect balanced chromosomal abnormalities;
however, studies have shown that some cases interpreted as
‘‘balanced’’ by conventional cytogenetics are in fact not
balanced. CMA can detect cryptic deletions or breakpoints in
40% of ‘‘balanced’’ cases; moreover, the true ‘‘balanced’’
chromosomal rearrangements account for 2% of cases, which
are unlikely to be related to the cause of a pregnancy loss
(Bruye
`re et al., 2002; Dhillon et al., 2014). If the CMA was
reported as normal, karyotyping did not detect any patho-
genic chromosomal imbalances. This result supports the idea
that CMA alone will not miss many significant results
(Rajcan-Separovic, 2012).
This study demonstrates that CMA can enhance the test
success rate and the resolution in the detection of unbalanced
genomic aberrations. Furthermore, SNP-based microarrays
can detect triploidy, LOH, uniparental isodisomy, and low-
level mosaicism. Therefore, genetic evaluation of POC is
central to the determination of the cause of pregnancy loss. As
more genetic mechanisms of pregnancy failure are elucidated,
unnecessary examinations and treatments will be reduced,
which will ultimately aid couples in the estimation of their
recurrence risk and in counseling for subsequent pregnancies.
Acknowledgments
This work was supported by the project of science
and technology of Guangdong Province (2014A020212354,
2013B051000087), Guangdong Medicine-Science Research
(A2015327), the National Natural Science Foundation of
China (31171229, U1132005), the Foundation for Ph.D. and
Returnees of Visiting Scholars of Guangzhou Medical Uni-
versity (2013C56), and the project of science and technology
of Guangzhou City (201400000004-4, 201400000003-4).
Author Disclosure Statement
No competing financial interests exist.
FIG. 3. SNP array analysis of POC samples 5, 6, and 7. (A, B, C) ChAS revealed a triploidy at four allele peaks in all of the
chromosomes (red arrow), whereas copy number signals could not infer any change in sample 5. (D) The purple bar of ChAS
indicated LOH in some regions. (E, F) ChAS showed a normal copy number, whereas the bottom row depicting the loss of a
heterozygous allele peak (red arrow) indicates an LOH region in sample 6. (G, H, I) The whole genome view shows a slightly
increased copy number signal in chromosome 13, indicating a mosaicism gain in sample. LOH, loss of heterozygosity. Color
images available online at www.liebertpub.com/gtmb
Table 3. SNP Array Results for POC Specimens from Different Stages of Pregnancy
Group Number
Abnormalities
(N/R)
Aneuploidy
and Triploidy
a
(N/R)
CNVs
(N/R)
Mosaicism
(N/R)
LOH
(N/R)
First trimester 22 12 (54.5%) 10 (45.5%) 1 (4.5%) 1 (4.5%) 0
Second or third
trimester
38 10 (26.3%) 5 (13.2%) 5 (13.2%) 0 1 (2.6%)
Total 60 22 (35%) 15 (25%) 6 (10%) 1 (1.7%) 1 (1.7%)
a
p<0.05
6 ZHANG ET AL.
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Address correspondence to:
XiaoFang Sun, MD
Key Laboratory for Reproduction and Genetics
of Guangdong Higher Education Institutes
Key Laboratory for Major Obstetric Diseases
of Guangdong Province
Third Affiliated Hospital of Guangzhou Medical University
Guangzhou 510150
China
E-mail: xiaofangsun@gzhmu.edu.cn
ChenHong Wang, MD
Shenzhen Maternity and Child Health Care Hospital
Shenzhen 518028
China
E-mail: wangchenhong@126.com
IMPLEMENTATION OF SNP ARRAY IN POC 7
