ORIGINAL ARTICLE Embryology
Effect of ICSI on gene expression
and development of mouse
G. Giritharan1, M.W. Li2, F. De Sebastiano1, F.J. Esteban3,
J.A. Horcajadas4, K.C.K. Lloyd2, A. Donjacour1, E. Maltepe1,
and P.F. Rinaudo1,*
1Department of Obstetric, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA
2Mouse Biology Program, Center for Comparative Medicine, University of California, Davis, CA 95618, USA3Department of Experimental
Biology, Systems Biology Unit, University of Jaen, Jaen 23071, Spain4Fundacion IVI and iGenomix, Valencia 46015, Spain
*Correspondence address. Division of Reproductive Endocrinology and Infertility University of California, San Francisco, 2356 Sutter St.,
San Francisco, CA 94115, USA. Tel: +1-415-353-7475; Fax: +1-415-353-7744; E-mail: firstname.lastname@example.org
Submitted on May 9, 2010; resubmitted on August 16, 2010; accepted on September 15, 2010
background: In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is
however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts.
methods: We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten’s medium
(ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo).
In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed
using the Affymetrix 430 2.0 chip.
results: Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with
embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; prolifer-
ation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes
was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa).
conclusions: Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the devel-
oping mouse embryo than the culture media used.
Key words: ICSI / embryos / microarray / gene expression / culture medium
It is estimated that 1% of children in the western world are born with
the help of assisted reproductive techniques (ART). Whereas these
techniques are thought to be safe, several studies report an increased
risk of unique complications associated with the use of IVF and ICSI
(Anthony, 2002; Cox et al., 2002; Hansen et al., 2002; Schieve
et al., 2002; DeBaun et al., 2003). The widespread use of ICSI, cur-
rently accounting for over 60% of the fertilization procedures in
USA (Society for Assisted Reproductive Technology, 2008 www.
sart.org) has sparked controversy because ICSI bypasses several
physiologic events which would usually occur during fertilization.
ICSI involves introduction of the sperm tail into the ooplasm and
may be associated with suboptimal sperm nuclear decondensation
(Markoulaki et al. 2007). In addition, ICSI-generated zygotes cleave
at a slower rate, have a reduced hatching rate and a reduced cell
number. ICSI zygotes also exhibit shorter calcium oscillations with
an altered pattern (Kurokawa and Fissore, 2003). Importantly,
Fernandez-Gonzalez et al. found that ICSI-generated mice using
fresh spermatozoa develop long-term consequences, such as obesity
and organomegaly, whereas ICSI-generated mice using frozen–
thawed spermatozoa had a more severe phenotype, including aber-
rant growth, abnormal behavior, early aging and increased incidence
of tumors (Fernandez-Gonzalez et al., 2008).
In our earlier studies, we showed that in vitro culture (IVC) of
zygotes to the blastocyst stage alters their global gene expression pat-
terns (Rinaudo and Schultz, 2004; Rinaudo et al., 2006). In addition,
IVF is associated with additional changes in global gene expression
and development (Giritharan et al., 2007). In order to investigate
the effect that ICSI has on mouse preimplantation embryo
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Human Reproduction, Vol.25, No.12 pp. 3012–3024, 2010
Advanced Access publication on October 1, 2010 doi:10.1093/humrep/deq266
development, and expand upon our prior IVC and IVF studies, we
carried out experiments fertilizing embryos by ICSI and culturing
embryos in different conditions, both optimal and suboptimal. Specifi-
cally, morphologic and gene expression studies were performed on
blastocysts fertilized by ICSI and grown in vitro either in Whitten’s
medium (WM) (ICSIWM group) or in KSOM with amino acids
(ICSIKSOMaagroup) compared with those fertilized and grown in vivo
(in vivo group). In addition, we compared the current data from ICSI
embryos cultured in WM to our previous data from IVF embryos cul-
tured in WM. Therefore, any comparisons between the IVFWMand
ICSIWMgroups would tease out the effects of gamete manipulation
using IVF and ICSI.
Materials and Methods
Collection of mature oocytes,
preimplantation mouse embryos
and ICSI procedure
Oocytes and embryos were isolated from super-ovulated dams as pre-
viously described (Rinaudo and Schultz, 2004). Briefly, CF-1 female mice
were injected with 5 IU pregnant mare’s serum gonadotrophin and
46–48 h later with 5 IU hCG. The following morning, oocytes were
obtained from the ampullae.Spermatozoa were collected from
B6D2F1/J male mice (10–11 weeks old). After mice were sacrificed,
the two caudal epididymides were removed, punctured gently with
30-G needles under a dissecting microscope and transferred with sterile
forceps into 1 ml pre-warmed (378C) EGTA Tris-HCl buffered solution
containing 10 mM Tris–HCl, pH 8.2, 50 mM EGTA and 50 mM NaCl in
a 1.5 ml microcentrifuge tube and then incubated at 378C for 10 min.
The top 0.8 ml sperm suspension was carefully aspirated and used
immediately for ICSI. Only oocytes with first polar body were microin-
jected using a Piezo drill (PMM Controller, Prime Tech, Ibaraki, Japan),
as described (Li et al., 2009). Then fertilized oocytes were washed and cul-
tured in either WM or KSOM with amino acids to the blastocyst stage
under 5% CO2in humidified air at 378C. Late-cavitating blastocysts of
similar morphology were harvested at ?112 h post-hCG for ICSI
groups and 96 h post-hCG for control in vivo group. Each treatment was
repeated four times. Each time, sperm from one male and oocytes from
two to three females were used for fertilization of ICSI groups. For in
vivo groups, eggs were collected from two to three females after overnight
breeding as one male per female. All animal experiments were approved
by the Institutional Animal Care and Use Committee.
Differential embryo staining
A modified dual nuclear staining method described by Thouas et al. (2001)
was performed to differentially stain inner cell mass (ICM) and trophoblast
cell nuclei of fully developed blastocysts. Briefly, the blastocysts were
exposed to 1% Triton X-100 (Sigma-Aldrich, USA) in minimum essential
medium (MEM; Invitrogen Corporation, USA) for 3–5 seconds, washed
3–5 times in MEM + polyvinylpyrrolidone (PVP; Sigma-Aldrich) and
incubated in MEM + PVP containing 100 mg/ml propidium iodide
(Sigma-Aldrich) for 30 seconds. The embryos were then washed 3–5
times in MEM + PVP and fixed overnight in 100% ethanol containing
25 mg/ml of bizbenzamide (Sigma-Aldrich) at 48C. The embryos were
mounted on a clean glass slide using glycerol and kept in a dark
chamber until observed under fluorescent light using a standard Leica
microscope (Model DMRB, Leica Microsystems AG, Germany).
The embryos were observed by fluorescent microscopy, and the
numbers of ICM cell (blue) and trophectoderm (TE: red) nuclei were
counted and photographed. The staining procedure was repeated at
least three times per treatment group with different sets of blastocysts
(n ≥ 12).
RNA extraction and amplification
Total RNA was extracted from four independent biological replicates (10
blastocysts/replicate) using PicoPure RNA Isoloation Kit (Arcturus, USA)
according to the manufacturer’s instructions. In column DNase treatment
was performed using RNase-free DNase set (Qiagen Inc., USA) as
described in the user guide of the PicoPure RNA isolation kit to remove
the residual DNA. In each pool, five embryo equivalents of total RNA
was used for reverse transcription (RT) followed by linear amplification
of antisense cDNA strand, fragmentation and biotin labeling by using
NuGEN Ovation Biotin System (NuGEN Technologies Inc., USA).
Briefly, RNA is converted to cDNA with a unique DNA/RNA heterodu-
plex at one end. A linear isothermal DNA amplification process was con-
ducted using DNA/RNA chimeric primer, DNA polymerase and RNase H
in a homogeneous isothermal assay that provides highly efficient amplifica-
tion of DNA sequences. The amplified cDNA strands were subjected to
an enzymatic fragmentation and the fragmented product was then labeled
with biotin. RNA and cDNA mass and size distribution were determined
before and after amplification, and after fragmentation using the Agilent
Bioanalyzer (Agilent, USA). Samples with RNA integrity number (RIN)
. 8 were selected for amplification. cDNA yield before fragmentation
and labeling was 12–15 mg. Final yield of fragmented and biotinylated
cDNA was 4–4.5 mg, of which 2.2 mg were used for microarray analysis.
Fragmented and biotin labeled cDNA samples were submitted to the
Genomic Core Facility of the University of California San Francisco for
GeneChip hybridization. The samples (four biological replicates per exper-
imental group) were hybridized to mouse Affymetrix 430 2.0 GeneChips,
then washed and stained on fluidics stations and scanned at 3 mm resol-
ution according to the manufacturer’s instructions (GeneChip Analysis
Technical Manual, www.affymetrix.com).
cDNA preparation for real-time PCR analysis
RT was accomplished by utilizing the commercially available first strand
cDNA synthesis kit (iScript cDNA Synthesis Kit, Bio-Rad Laboratories,
USA). The RT reactions were performed by following the kit manufac-
turer’s protocol. For each treatment group the RT was repeated four
times with RNA from different sets of blastocyst stage embryos.
The real-time PCR was performed using TaqMan Universal PCR Master
Mix, Gene Expression Assays containing gene-specific primers and
TaqMan probe (Applied Biosystems, USA), and 0.1 embryo equivalents
of cDNA. The corresponding ABI TaqMan Assay-on-Demand probe/
primer sets used were Mm00498012_m1 (Ube2a), Mm00438084_m1
(Ccng1), Mm99999915_g1 (Gapdh) and Mm00433832_m1 (Nr3c1). The
real-time PCR was also performed using SyBr green PCR supermix
(Bio-Rad Laboratories), and primers for Bdnf, Nr3c1 and Gapdh genes
with 0.1 embryo equivalents of cDNA from each treatment group.
Primers which span at least two exons were designed using Primer3 soft-
ware. Duplicates were used for each real-time PCR reaction; a minus tem-
plate served as control. For each treatment group the real-time PCR was
repeated four times. The real-time PCR data were analyzed within the log-
linear phase of the amplification curve obtained for each probe/primer
using the comparative threshold cycle method (Bio-Rad Laboratories).
Effect of ICSI on mouse preimplantation embryo
The cell number data were analyzed by one-way analysis of variance. Cell
numbers were compared using a mean separation procedure when analy-
sis of variance showed significant F-values using Fisher’s Least Significant
Difference method. Results are reported as the mean values for each
set of data + SEM.
Microarray gene expression data analysis
Statistical analysis was carried out using the R software (http://www.
r-project.org/) and the appropriate Bioconductor packages (http://
www.bioconductor.org/) run under R. In order to remove all the possible
sources of variation of a non-biological origin between arrays, densitome-
try values between arrays were normalized using the RMA (robust multi-
array) normalization function implemented in the Bioconductor affylmGUI.
Statistically significant differences between groups were identified using the
rank product non-parametric test implemented in the Bioconductor Rank-
Prod package. Applying a Student’s t-test with such a limited number of
samples (four in each experimental group) is inappropriate as the obtained
statistical significance is not robust; in this situation the mean and the stan-
dard deviation could be easily biased by outliers. We therefore carried out
a non-parametric statistical test as a rough filter to narrow down the list of
most relevant genes (Breitling and Herzyk, 2005). This non-parametric
method is highly efficient, robust and widely used for microarray data
analysis (Cervello et al., 2010). The Rank Product method has proven
to be superior to other statistical methods for microarray data analysis
in our and other authors’ experience (Hong and Breitling, 2008). More-
over, the rank product approach includes a multiple hypothesis test for
raw P-value correction to ascertain a false positive rate similar to false dis-
covery rate correction. Data of the microarray are available at the Gene
Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo).
Genes flagged as ‘present’ or ‘marginal’ in at least one hybridization,
based on raw perfect match and mismatch as determined by ‘mas5calls’
function on the Bioconductor affymetrix package, were considered
Functional annotations were carried out using the Ingenuity Pathways
Analysis platform (http://www.ingenuity.com/).
Validation of microarray data using
Published evidence confirms that microarray data are robust and
reliable (Morey et al., 2006). In order to evaluate the reproducibility
of the data, we performed multiple quality control analysis. Initially,
we only utilized expanded blastocysts of similar morphology and we
performed the experiments using four independent biological repli-
cates. We confirmed that the quality of RNA was optimal, utilizing
only RNA samples with RIN . 8. We then performed in silico
quality control analysis of the amplified material: we found that
cRNA quality was excellent. Finally, we utilized real-time RT–PCR
to confirm the microarray results obtained in this study (Fig. 1). In par-
ticular, we chose one gene with increased expression in the ICSI group
(Ccgn1), one gene with decreased expression (Ube2a) and two genes
with no change in expression between ICSI and in vivo blastocysts
(Bdnf and Nr3c1). Results confirm similar trend between microarray
and real-time data. The only difference is represented by the statisti-
cally significant (P , 0.05) increase after RT–PCR in Nr3c1 expression
in ICSIWM blastocysts, although the increase of Nr3c1 following
microarray analysis in both media or RT–PCR in ICSIKSOMaais not
Effect of ICSI and the culture medium
on blastocyst cell number
Expanded mouse blastocysts are easily recognizable and therefore
offer an excellent end-point for morphologic assessment. In order
to investigate the effect of the method of fertilization and IVC on
cell division and cell allocation, we counted the number of ICM and
TE cell nuclei in expanded blastocysts. Only expanded blastocysts at
the same stage of development were used for analysis (Table I).
In vivo embryos had on average two additional ICM cells compared
with ICSIKSOMaa and three additional ICM cells compared with
ICSIWMembryos (P , 0.05). The ICSIWMand IVFWMembryos had
reduced number of TE cells (n ¼ 36 and 33 cells, respectively) com-
pared with the ICSIKSOMaa (n ¼ 43 cells) and the in vivo control
Figure 1 Real-time PCR verification of gene expression data.
Expression profile of brain derived neurotrophic factor (Bdnf), cyclin
G1 (Ccng1), ubiquitin-conjugating enzyme E2A (Ube2a) and the
nuclear receptor subfamily 3, group C, member 1 (Nr3c1) genes in
murine blastocysts produced by in vivo fertilization and culture
(in vivo), ICSI and IVC in KSOM with amino acids (ICSIKSOMaa) and
ICSI and IVC in WM (ICSIWM) using real-time PCR technique (A)
and microarray technique (B). Bars represent SD. Asterisk (*) indi-
cates significant difference compare with in vivo at P , 0.05. Of
note the genes tested by RT–PCR reflect the changes detected by
microarray. The only difference is represented by the statistically sig-
nificant (P , 0.05) increase after RT–PCR in Nr3c1 expression in
ICSIWMblastocysts, while the increase of Nr3c1 following microarray
analysis in both media or after RT–PCR in ICSIKSOMaais not statisti-
Giritharan et al.
embryos (n ¼ 49, P , 0.05). Of note, the development of embryos
after ICSI, was excellent. More than 70% of the injected eggs survived
and more than 90% of the surviving eggs reached the 2-cell stage in
both culture media. The blastocyst development rates for 2-cell
embryos cultured in ICSIKSOMand ICSIWM were 80.7 and 76.4%,
Effect of ICSI and in vitro embryo culture
on blastocyst global gene expression
To analyze the global pattern of gene expression we used the mouse
Affymetrix 430 2.0 microarray chip, which is believed to represent the
complete mouse genome. Out of 12 035 total non-redundant genes
with known gene symbols, 8847 (73.5%) were present in ICSIWM,
8408 (69.8%) in ICSIKSOMaaand 7758 (64.4%) in in vivo blastocysts.
Pair-wise comparison revealed that a significant number of genes are
differentially regulated following ICSIWM(947) or ICSIKSOMaa(1016)
compared with in vivo control embryos (Fig. 2, P , 0.05). Most of
the differentially regulated genes between the ICSI groups and in vivo
are common (ICSIKSOMaa: 78.9%—802/1016; ICSIWM: 84.6%—802/
947), with only 215 genes being uniquely different between ICSIKSOMaa
and in vivo and 145 between ICSIWMand in vivo. This finding is impor-
tant because it provides additional evidence of the robustness of the
data. Indeed, unsupervised hierarchical clustering analysis confirms
these findings (Fig. 3). The samples segregated into two major cluster-
ing branches (ICSI and in vivo). However, all the replicate samples of
the ICSI embryos cultured in WM and KSOMaa did not show a
clear separation, indicating that the ICSI procedure itself affects the
transcriptome of the blastocyst and that the culture media plays
only a minor role.
The entity of fold changes was higher than expected: 20.6% (210/
1016) of the genes were changed more than 5-folds between
ICSIKSOMaaand in vivo, and 20.4% (194/947) were different between
ICSIWMand in vivo blastocysts.
Remarkably, a reduced number of genes were differentially
expressed in ICSIKSOMaaembryos compared with ICSIWM(41 genes,
P , 0.05); in addition only three genes were changed more than
3-fold between the two groups, indicating a minor effect of culture
on gene expression following ICSI procedure.
The list of significantly different genes after each pair-wise compari-
son with the relative expression profiles is summarized in Supplemen-
tary data, Table S1.
Pathways analysis (Table II) reveals that similar classes are altered in
embryos generated by ICSI and in vivo, independently of the culture
media used. In particular there is an increase in alteration of pathways
oxidative phosphorilation and mitochondrial dysfunction pathways)
and alteration of metabolic pathways (N glycan degradation,
branch chained amino acids and butanoate metabolism). Only the
hepatocytes growth factor (HGF) signaling pathway and glycolysis/
gluconeogenesis pathway are uniquely different between ICSIKSOMaa
and in vivo, while several classes are uniquely changed between ICSIWM
and in vivo [Oct4-dependent pathway; production of reactive oxygen
species (ROS) in macrophages; CDK5 signaling; TR/RXR Activation].
Functional categories analysis (Fig. 4 and Supplementary data, Table
S2) shows that cell function, development and metabolic genes are
affected by the method of fertilization and culture media used.
Whereas all conditions used affect these functions, it is clear that
the method of fertilization plays a more important role in altering
the gene expression pattern than the media used to culture embryos.
In fact, only 41 genes and two pathways (one-carbon pool by folate
and RhoA signaling pathways) were different between ICSIKSOMaaand
ICSIWM, while ?1000 genes were different between ICSI embryos
and in vivo embryos.
Effect of the method of fertilization
(IVF or ICSI) on gene expression
Comparison of ICSIWM data with our previously generated gene
expression data-IVFWM(Giritharan et al., 2007) showed differential
expression of 981 genes. Eighteen percent (177/981) of these
genes were changed more than 5-fold.
Overall multiple functional categories involved in cell function,
development and metabolism (Supplementary data, Table S2) and
pathways (Table II) are statistically different between IVFWMand
Analysis of transporter genes (Table III) and imprinted and methyl-
ation genes (Table IV) reveals that several genes are affected by the
method of fertilization.
This study, for the first time, offers a complete overview of the
changes in the global pattern of gene expression in ICSI or in vivo gen-
erated mouse embryos. This provides an overview of the transcrip-
tional consequences of fertilizing eggs by different techniques and
complements our previous work on the effect of different media
and oxygen concentration on the transcriptome of mouse blastocysts
(Rinaudo and Schultz, 2004; Rinaudo et al., 2006; Giritharan et al.,
2007). The most notable finding is that the ICSI procedure plays a
more important role in determining the blastocyst gene expression
pattern than the culture media used (WM or KSOMaa).
ICSIKSOMaa(n ¼ 31)
ICSIWM(n ¼ 12)
IVFWM(n ¼ 63)
In vivo (n ¼ 46)
Table I The method of fertilization affects the distribution of cells in ICM and TE of embryos.
Treatment# ICM cells (mean+++++SEM)# TE cells (mean+++++SEM)# Total cells (mean+++++SEM)
Numbers are expressed as means+SEM. Values with different superscripts in each column differ significantly (P , 0.05).
Effect of ICSI on mouse preimplantation embryo
Overall, there is a net reduction of ICM and TE cells in the ICSI
group compared with the in vivo control. The reduction of cells is
larger in TE cells (?26% less cells in ICSIWMand 14% in ICSIKSOMaa)
than ICM (?10% less in ICSI embryos) of ICSI embryos. Since TE cells
will give rise to the placenta, this suggests that the method of fertiliza-
tion could affect more placenta formation than the embryo itself. The
cell number data confirm findings of other investigators, who found
that, in various species, ICSI-generated zygotes cleave at a slower
rate and have reduced hatching rate and reduced cell number
(Dumoulin et al., 2000; Bedford et al., 2003; Malcuit et al., 2006).
Although it is not possible to compare the present mouse findings
with human data, it is interesting to note that lower hCG levels were
found in human IVF gestations than in in vivo conception, implying
reduced placental mass (Zegers-Hochschild et al., 1994). Assuming
that an embryo with lower cell numbers will grow less, these findings
could explain the fact that ART children have lower birthweight at
term (McDonald et al., 2009).
The gene expression results confirm the morphologic studies. The
ICSI embryos have a very distinct transcriptome compared with
embryos fertilized in vivo, as shown by the unsupervised hierarchical
clustering analysis (Fig. 3). ICSI embryos have an increased dysregula-
tion (both up- and down-regulation) in the expression of genes related
to cellular function, development and metabolism. The down-
regulation of several genes involved in cellular development and differ-
entiation (Fig. 4 and Supplementary data, Table S2) implies that an
alteration of developmental strategy could follow the preimplantation
stress and explains the reduced number of cells in ICSI embryos.
Overall, the increased number of dysregulated genes indicates that
these embryos have an increased metabolism; this would suggest
decreased fitness according to the ‘quiet embryo’ hypothesis (Leese
et al., 2008).
Pathway analysis offers additional insights (Table II): mitochondrial
dysfunction and a disproportionately higher number of metabolic path-
ways occurring in mitochondria (inositol, butanoate, urea cycle and
branched amino acid metabolism) are altered following ICSI. Individual
gene analysis confirms these findings. For example, Cox6b2 (cyto-
chrome c oxidase subunit VIb polypeptide 2) gene is increased
more than 20-fold in ICSI embryos. This gene produces an isoform
of cytochrome c oxidase, the terminal enzyme in the mitochondrial
respiratory chain, which catalyzes the electron transfer from
reduced cytochrome c to molecular oxygen (Huttemann et al.,
2003). Up-regulation of mitochondrial oxidative phosphorylation
genes indicates the increased metabolic need of the embryo.
Of note, mitochondrial dysfunction can result in reduced post-
implantation development (Thouas et al., 2006) and can have long-
lasting effects. For example, it has been hypothesized as the mechan-
ism inducing the insulin-resistant phenotype observed in offspring of
patients with type 2 diabetes (Petersen et al., 2004).
Among the signaling pathways modified after ICSI, the g amino
butyric (GABA) receptor signaling and invasiveness signaling are pro-
minent. Autocrine and paracrine GABA signaling via GABAAreceptors
slow preimplantation embryonic growth by decreasing proliferation
Figure 2 Summary of up-regulated and down-regulated genes for the following comparisons: ICSIKSOMaaversus in vivo, ICSIWMversus in vivo,
ICSIKSOMaaversus ICSIWM. Each comparison in the graph shows the number of genes statistically different when comparing the four replicates of
one group with the four replicates of the other group. The numbers below each comparison indicate how many genes are statistically up-regulated
or down-regulated (P , 0.05) more than 2-fold or 5-fold.
Giritharan et al.
because of increased cellular arrest in the S phase (Andang et al.,
2008). Alteration of invasiveness signaling could indicate suboptimal
placentation. For example, Timp1, an inhibitor of cellular invasion
(Paiva et al., 2009) and Itgb5 (integrin beta 5), an integrin involved in
implantation (Massuto et al.) are up-regulated in ICSI placentas.
Alteration of multiple additional genes involved in placentation is
present. This finding provides a molecular justification of the significant
reduction in TE cells found in ICSI embryos. Prl2c2 (prolactin family 2,
subfamily c, member 2) is expressed in large trophoblastic giant cells
during the invasion (Adamson et al., 2002) and it is down-regulated
25-fold in ICSI embryos. Psg28 (pregnancy-specific glycoprotein 28)
is expressed in giant cells and spongiotrophoblast of the placenta
(Kromer et al., 1996), is implicated in immunomodulatory function
to prevent rejection of embryo/fetus (Wynne et al., 2006) and is
down-regulated more than 11-fold in ICSI blastocysts. Bex1
(brain-expressed X-linked 1) located close to Xist on the X chromo-
some, is down-regulated more than 16 times in ICSI embryos; while it
does not appear to be epigenetically regulated, it is highly expressed in
trophectodermal cells (Williams et al., 2002). Down-regulation of
these genes could be associated with reduced placental development.
Alteration of multiple cellular transporters could indicate the pla-
centa function is compromised. Among the transporter genes
(Table III), Slc7a12 (solute carrier family 7, member 12) stands out
for being down-regulated more than 18-fold in ICSI embryos; this
gene is involved in the cationic amino acid transport (Closs et al.,
2006). Dysregulation of other transporter genes has been associated
with different metabolic or storage diseases. Although so far none
of the following diseases have been linked to ART, alterations of
these genes implies metabolic stress. Slc35c1 (solute carrier family
35, member C1) encodes a GDP-fucose transporter located in the
Golgi apparatus. Mutations in this gene result in congenital disorder
of glycosylation type IIc (Lubke et al., 2001). Slc39a4 (solute carrier
family 39, member 4) encodes for a transmembrane protein required
for zinc uptake in the intestine. Mutations in this gene result in acro-
dermatitis enteropathica, a rare inherited defect in the absorption of
dietary zinc (Wang et al., 2002). Slc46a1 (solute carrier family 46
member 1) is a transmembrane proton-coupled folate transporter
that facilitates the movement of folate and heme across membranes.
Mutations in this gene cause a recessive form of folate malabsorption
(Qiu et al., 2006).
Because abnormal DNA methylation can follow culture in vitro, we
paid particular attention to imprinting and methylation genes (Doherty
et al., 2000; Rolaki et al., 2007). Several imprinted genes are different
between ICSI embryos and in vivo embryos (Table IV). Among them
Cd81 and Peg10 are changed more than 10-fold. Cd81 encodes a tet-
raspanin that functions as an organizer of multi-molecular membrane
complexes and as a result, regulates cell migration, fusion and signaling
(Hemler, 2005). Cd81 is located close to the cluster of imprinted
genes on murine chromosome 7 which is syntenic with the Beck-
with–Wiedemann syndrome associated cluster of imprinted gene
on human chromosome 11p15.5 region (Paulsen et al., 1998).
Lim et al. (2009) reported that children born after ICSI with
Figure 3 Hierarchical clustering with heat-map of ICSIKSOMaa, ICSIWMand in vivo embryos based on their gene expression profile. Unsupervised
clustering was performed to analyze similarities among replicate samples across all treatment groups tested. Colors correspond to relative RNA abun-
dance for the transcripts detected; each is represented by a horizontal bar in the heat-map. Yellow indicates high expression and blue denotes low
Effect of ICSI on mouse preimplantation embryo
Altered pathways with fold change in ICSI embryos (both ICSIWMand ICSIKSOM) compared with in vivo embryos
GABA receptor signaling0.109Abat (4.6, 3.6), Gabra5 ( 23.1, 23.3), Gabrb2 (22.7, 22.5), Gabrg1 (22.2, 22.3), Slc6a13
Invasiveness signaling (Glioma)0.107F2r (3.6, 5.3), Itgb5 (4.2, 4.5), Rhod (5.3, 4.4), Rhoj (22.7, 22.7), Rras (3.7, 3.9), Timp1 (4.8,
Inositol metabolism0.143 Adi1 (22.5, 22.5), Aldh16a1 (3.3, 3.7), Blvrb (9.5, 11.1), Cyba (6.5, 8.5), Dhrs1 (5.4, 4.3),
Dhrs13(3.7, 3.7),Lamb2(4.7,4.5), Ndor1(4.1,4.1),Ndufa6 (3.6,3.4),Recql4 (3.7,3.5),Retsat
(3.3, 3.5), Tm7sf2 (3.9, 3.6)
Butanoate metabolism0.133Abat (4.6, 3.6), Aldh2 (3.5, 3.5), Dcxr (14.3, 15.1), Ech1 (4.4, 5.7), Hadha (4.0, 4.1), Nlgn1
(22.6, 22.8), Oxct1 (3.9, 4.1), Sdhb (3.8, 4.0)
N-glycan degradation0.133 Man1b1 (4.1, 3.4), Man1c1 (3.1, 4.2), Man2c1 (3.8, 4.2), Manea (3.2, 3.5)
Urea cycle and metabolism of amino groups0.118Acy3 (6.1, 4.9), Ass1 (4.1, 3.7), Oat (3.4, 4.0), Pycrl (4.0, 3.4)
Valine, leucine and isoleucine degradation0.101Abat (4.6, 3.6), Aldh2 (3.5, 3.5), Bcat2 (4.7, 5.4), Bckdhb (4.4, 4.5), Ech1 (4.4, 5.7), Hadha (4.0,
4.1), Oxct1 (3.9, 4.1)
Glutathione metabolism0.094 Gpx3 (8.0, 7.7), Gpx7 (5.3, 6.2), Gstk1 (5.8, 4.7), Gstm2 (10.6, 13.4), Mgst3 (4.3, 3.9), Oplah
Ubiquinone biosynthesis0.127Bckdhb (4.4, 4.5), Ndufa1 (4.0, 3.9), Ndufa13 (3.8, 3.6), Ndufa3 (4.1, 4.2), Ndufa6 (3.6, 3.4),
Ndufb3 (3.8, 4.0), Ndufb7 (4.1, 4.0), Ndufs7 (4.4, 4.2), Ndufs8 (3.3, 3.3)
Oxidative phosphorylation0.105Atp5e (3.4, 3.7), Atp5g2 (3.2, 3.9), Cox6b2 (22.9, 24.9), Cox7a1 (7.0, 5.8), Ndufa1 (4.0, 3.9),
Ndufa13 (3.8, 3.6), Ndufa3 (4.1, 4.2), Ndufa6 (3.6, 3.4), Ndufb3 (3.8, 4.0), Ndufb7 (4.1, 4.0),
Ndufs7 (4.4, 4.2), Ndufs8 (3.3, 3.3), Ppa2 (6.6, 7.7), Sdhb (3.8, 4.0), Uqcr (4.1, 4.2)
Mitochondrial dysfunction0.092Cox6b2 (22.9, 24.9), Cox7a1 (7.0, 5.8), Gpx7 (5.3, 6.2), Ndufa13 (3.8, 3.6), Ndufa3 (4.1, 4.2),
Ndufa6 (3.6, 3.4), Ndufb3 (3.8, 4.0), Ndufb7 (4.1, 4.0), Ndufs7 (4.4, 4.2), Ndufs8 (3.3, 3.3),
Sdhb (3.8, 4.0), Snca (22.6, 22.6)
Pathways uniquely changed between ICSIWMand in vivo embryos
Role of Oct4 in mammalian embryonic stem cell
CDK5 signaling0.034Egr1 (22.6), Ppp1r11 (3.3), Ppp1r3a (22.5)
TR/RXR activation0.033Ppargc1a (22.2), Rxrb (3.5), Ucp2 (3.2)
Production of nitric oxide and reactive oxygen
species in macrophages
Pathways uniquely changed between ICSIKSOMand in vivo embryos
HGF signaling0.04Ccnd1 (22.2), Elf3 (3.4), Elf5 (3.7), Map3k6 (3.5)
Glycolysis/gluconeogenesis 0.042 Aldh3b2 (3.4), Galm (3.2), Ldha (4.0), Pfkm (3.7)
Pathways uniquely changed between ICSIKSOMand ICSIWMembryos
RhoA signaling pathways0.019 Myl9 (2.05), Sept4 (2.04)
One-carbon pool by folate 0.048 Mthfd2 (2.33)
Pathways changed between IVFWMand ICSIWM
Invasiveness signaling (Glioma)0.125 F2r (27.87), Itgb5 (23.41), Rhod (23.28), Rhoj (2.35), Rnd2 (22.86), Rras (24.83), Timp1
Table II Overrepresented canonical pathways in comparisons among ICSIKSOM, ICSIWMand in vivo embryos (P < 0.05).
Ingenuity canonical pathwaysRatio Gene symbol
0.044Fgf4 (22.3), Rxrb (3.5)
0.031 Map3k4 (3.7), Mapk13 (3.9), Ppp1r11 (3.3), Ppp1r3a (22.5), Rnd2 (3.8)
Giritharan et al.
Beckwith–Wiedemann syndrome have an altered methylation in this
region of chromosome 11. It is, however, important to remember
that this gene is imprinted in mice but not humans and therefore con-
clusions valid in one model system may not be valid in another species.
Peg10 (Paternally expressed 10) is an imprinted gene down-regulated
more than 18-fold in ICSI embryos; it plays an important role in pla-
cental development and its deletion is associated with early embryonic
lethality (Rawn and Cross, 2008).
Interestingly, additional genes that are not imprinted but are located
on the X chromosome and therefore potentially subjected to selective
epigenetic controls were altered. Among these, Ott (ovary testis tran-
scribed) is a mouse X-linked multigene family gene specially expressed
during meiosis in testis and ovary (Kerr et al., 1996), while Slc10a3
(solute carrier family 10 member 3) belongs to the sodium/bile acid
cotransporter family and was originally cloned from placental tissue;
it maps to a GC-rich region of the X chromosome and was identified
by its proximity to a CpG island (Geyer et al., 2006).
Several genes involved in epigenetic regulation appear to be misre-
gulated. Importantly Wbp7, Smarca1 and Hdac6 are altered in ICSI
Hdac6 belongs to the histone deacetylases (HDACs) family of
enzymes that catalyze the removal of acetyl groups from lysine resi-
dues in histones and non-histone proteins, resulting in transcriptional
repression. HDACs play a role in cell growth arrest, differentiation
and death, and Hdac6 over expression has been associated with pre-
mature chromatin compaction in mouse oocytes and embryos (Verdel
et al., 2003).
Smarca1 (SWI/SNF related, matrix associated, actin-dependent
regulator of chromatin, subfamily a, member 1) encoded protein has
helicase and ATPase activity and regulates transcription by altering
chromatin structure (Magnani and Cabot, 2009).
Wbp7 (WW domain binding protein 7) is a histone methyltransfer-
ase that methylates Lysine 4 of histone H3 resulting in an epigenetic
mark favoring transcriptional activation. Mice lacking Wbp7 fail to
develop beyond E9.5 (Lubitz et al., 2007).
Urea cycle and metabolism of amino groups 0.176Acy3 (23.30), Aldh18a1 (25.21), Cps1 (3.72), Oat (23.77), Pycrl (23.31), Srm (24.52)
Glutathione metabolism 0.125Ggt1 (23.80), Gpx4 (23.72), Gpx7 (24.64), Gstk1 (23.40), Gstm2 (25.77), Mgst1 (2.19),
Mgst3 (23.05), Oplah (25.19)
Phospholipid degradation 0.103 Dgka (23.23), Hmox1 (23.99), Lamb2 (23.89), Napepld (23.84), Pla1a (23.25), Pla2g10
(22.96), Plcd1 (25.23), Pld3 (23.05), Sphk2 (23.39)
Selenoamino acid metabolism0.132Cbs (25.85), Cth (25.93), Edf1 (23.16), Ggt1 (23.80), Mars2 (23.00)
Glycerophospholipid metabolism0.092Agpat2 (23.34), Dgka (23.23), Hmox1 (23.99), Lamb2 (23.89), Napepld (23.84), Pcyt2
(24.84), Pemt (22.59), Pla1a (23.25), Pla2g10 (22.96), Plcd1 (25.23), Pld3 (23.05),
Glutamate metabolism 0.132Abat (25.49), Cad (24.01), Cps1 (3.72), Nadsyn1 (22.47), Tgm4 (23.55)
Inositol metabolism0.131 Adi1 (2.20), Aldh16a1 (23.80), Blvrb (26.23), Cyba (27.99), Dhrs1 (24.05), Dhrs13
(23.73), Hmox1 (23.99), Lamb2 (23.89), Ndor1 (23.62), Recql4 (24.66), Tm7sf2 (24.01)
Pyrimidine metabolism0.111Apobec1 (2.38), Cad (24.01), Dhodh (24.03), Mad2l2 (23.24), Nme3 (25.20), Nme4
(23.70), Nt5m (23.21), Pold1 (24.68), Pold2 (29.11), Pold4 (23.58), Poli (23.79), Polr2i
(25.06), Polr2l (25.06), Polrmt (23.35), Trub2 (23.57), Uckl1 (23.20), Upp1 (23.39)
Mitochondrial dysfunction 0.115Cox6b2 (214.50), Cox7a1 (25.05), Cox7b2 (22.33), Dhodh (24.03), Gpx4 (23.72), Gpx7
(24.64), Hsd17b10 (23.24), Ndufa3 (24.04), Ndufb3 (23.61), Ndufb7 (23.25), Ndufs2
(23.71), Ndufs7 (23.88), Ndufs8 (23.32), Park7 (23.28), Ucp2 (23.22)
Oxidative phosphorylation0.091Atp5e (23.69), Cox6b2 (214.50), Cox7a1 (25.05), Cox7b2 (22.33), Ndufa1 (23.10),
(23.32), Ppa2 (25.10), Uqcr (24.16)
Ubiquinone biosynthesis0.127 Bckdhb (24.41), Edf1 (23.16), Ndufa1 (23.10), Ndufa3 (24.04), Ndufb3 (23.61), Ndufb7
(23.25), Ndufs2 (23.71), Ndufs7 (23.88), Ndufs8 (23.32)
Table II Continued
Ingenuity canonical pathwaysRatio Gene symbol
Figure 4 A total number of differentially expressed genes in cellu-
lar, developmental and metabolic functional catergories overrepre-
sented in ICSIKSOMaa, ICSIWM, and in vivo embryos (P , 0.05).
Detail information of cellular, developmental and metabolic functions
with differentially regulated genes in each comparison is listed in Sup-
plementary data, Table S2.
Effect of ICSI on mouse preimplantation embryo
The transcriptome comparison of ICSI embryos cultured in KSOMaa
or WM and the comparison of ICSIWMand IVFWMblastocysts pro-
vided some of the most unanticipated findings of the study: the
method of fertilization plays a more important role than the culture
media to determine the transcriptome of the blastocysts.
Regarding the comparison of ICSIKSOMaaand ICSIWM, it appears
that while ICSIWMembryos have a statistically reduced number of
TE cells (18% less), only 41 genes (4%—versus ?1000 genes different
between in vivo and ICSI blastocysts) were differentially expressed; in
addition the fold change differences were small and overall ,4-fold.
Interestingly, H19 is the only imprinted gene differentially expressed
being up-regulated more than 3-fold in WM. This is not surprising,
since culture in WM, as opposite to culture in KSOM, is associated
with biallelic expression of the gene (Doherty et al., 2000).
Only three transporter genes are differentially regulated (Slc44a3;
Finally only two pathways were statistically different between
ICSIKSOMaaand ICSIWM(RhoA signaling and one carbon folate path-
ways). Members of the Rho family of small guanosine triphosphatases
have emerged as key regulators of the actin cytoskeleton, and further-
more, through their interaction with multiple target proteins, they
ensure coordinated control of other cellular activities such as gene tran-
scription and adhesion. Down-regulation of Mthfd2 (methylenetetrahy-
Slc10a3Sodium/bile acid cotransporter
Slc12a4 Potassium/chloride transporters
Slc13a2Sodium-dependent dicarboxylate transporter
Slc16a6Monocarboxylic acid transporter
Slc22a9Organic anion transporter
Slc25a28Mitochondrial iron transporter
Slc27a3Fatty acid transporter
Slc35c1 GDP-fucose transporter
Slc39a11Metal ion transporter
Slc3a2Activators of dibasic and neutral amino acid transport
Slc44a4 Choline transporter
Slc5a6 Sodium-dependent vitamin transporter
Neurotransmitter (GABA) transporter
Slc7a12Cationic amino acid transporter
Slc7a13 Cationic amino acid transporter
Slco6b1Organic anion transporter
Slco6c1 Organic anion transporter
Slc27a2Fatty acid transporter
Slc27a4Fatty acid transporter
Slc38a7Sodium-coupled neutral amino acid transporter
Slc39a12 Zinc transporter
Slc4a2 Anion exchanger
Slc5a6Sodium-dependent vitamin transporter
Slc7a3 Cationic amino acid transporter
Slco6d1 Organic anion transporter family
Table III Differentially expressed transporter genes in ICSI and IVF embryos (P < 0.05).
NS, not significant.
Giritharan et al.
notable as this gene is involved in folate metabolism and therefore could
result in abnormal methyl donor availability for methyltransferases.
Complementary to the findings of ICSI embryos cultured in different
media, the different gene expression pattern between IVFWMand
ICSIWMconfirms that the method of fertilization plays a fundamental
role in determining the transcriptome of the preimplantation
embryos. In fact, as many genes are different between IVFWMand
ICSIWM (984 gene) as between ICSIKSOMaa and in vivo (1016) or
ICSIWMand in vivo (947).
Pathways analysis confirms that similar pathways (Mitochondrial
function pathways and metabolic pathways) are changed between
IVFWMblastocysts and ICSIWMembryos as between ICSI embryos
(both WM and KSOMaa) and in vivo embryos.
Interestingly, among the transporters differentially expressed, a
subset is uniquely mis-expressed between IVFWMand ICSIWMblasto-
cysts (Table III). Slc27a4 is involved in translocation of long-chain fatty
acids across the plasma membrane and is required for fat absorption in
early embryogenesis (Gimeno et al., 2003). Slc37a4 (solute carrier
family 37 member 4) regulates glucose-6-phosphate transport from
the cytoplasm to the endoplasmic reticulum and is involved in ATP-
mediated calcium sequestration in the lumen of the endoplasmic reti-
culum. Mutations in this gene have been associated with various forms
of glycogen storage disease (Schaub and Heyne, 1983).
Three epigenetic genes (Wbp7, Kcnq1 and Smarca4) were differen-
tially regulated in IVFWMand ICSIWMembryos. Kcnq1 was down-
regulated in ICSI embryos while Wbp7 and Smarca4 were up-regulated
in ICSI embryos compared with IVF embryos. Smarca4 protein plays a
fundamental roles controlling gene expression during early mammalian
embryogenesis (Magnani and Cabot, 2009). Wbp7 also plays a key
role in embryo development, and regulates the apoptosis and differen-
tiation of cells into all three germ layers (Lubitz et al., 2007). Most
recent evidence revealed an association of the Kcnq1 gene with the
susceptibility to type 2 diabetes. Kcnq1 participates in the regulation
of cell volume, which is, in turn, critically important for the regulation
of metabolism by insulin. Kcnq1 counteracts the stimulation of cellular
K+ uptake by insulin and thereby influences K+-dependent insulin
signaling on glucose metabolism (Boini et al., 2009).
Importantly, this study does not address unequivocally whether the
differences in gene expression described in ICSI-produced blastocysts
are secondary to the ICSI procedure alone or the combination of ICSI
and IVC. An ideal experiment would include the transcriptome analy-
sis of blastocysts that were generated by ICSI, with the resulting two
pronuclei embryos immediately transferred to recipients to eliminate
the effects of in vivo culture. Whereas this experiment is feasible, it
is technically difficult and requires multiple manipulations. The next
best experiment is to observe the gene expression changes of
zygote generated in vivo and cultured to the blastocyst stage (IVC
embryos). We (Rinaudo and Schultz, 2004; Giritharan et al., 2007)
and others (Wang et al., 2005; Zeng and Schultz, 2005; Fernandez-
Gonzalez et al. 2009) have shown that culture conditions play an
important role in shaping gene expression in developing embryos
and blastocysts. In particular, in one study we showed that culture
media and oxygen concentration play an independent role in affecting
gene expression, with the oxygen concentration determining a syner-
gistic increase in gene misregulation. IVC of zygotes in atmospheric
oxygen (20% O2) was associated with markedly increasing misregu-
lated genes (20% O2:WM: 354 genes changed more than 2-fold com-
pared with in vivo flushed blastocysts; KSOMaa: 102 differentially
expressed gene), compared to IVC of zygotes in physiologic oxygen
concentration (5% O2: WM: 45 genes and KSOM 18 genes changed
more than 2-fold, respectively; Rinaudo et al., 2006). In a separate
study, we showed that a high number of transcripts was statistically
Asb4Ankyrin repeat and SOCS box-containing 4
Cd81 CD81 antigen
H19H19 fetal liver mRNA
Igf2as Insulin-like growth factor 2, antisense
Kcnq1Potassium voltage-gated channel, subfamily Q, member 1
Osbpl5 Oxysterol binding protein-like 5
Peg10Paternally expressed 10
Epigenetic regulating genes
Hdac6 Histone deacetylase 6
Smarca1SWI/SNF related, matrix associated, actin-dependent regulator
of chromatin, subfamily a, member 1
Wbp7WW domain binding protein 7
Mbd2 Methyl-CpG binding domain protein 2
Smarca4 SWI/SNF related, matrix associated, actin-dependent regulator
of chromatin, subfamily a, member 4
Table IV Imprintedand epigenetic genes differentiallyexpressed in ICSIand IVFembryos compared within vivoembryos
with their fold changes (P < 0.05).
NS, not significant.
Effect of ICSI on mouse preimplantation embryo
different in blastocysts generated by IVF and cultured in WM 20% O2
when compared with in vivo control blastocysts, but the magnitude of
the changes in gene expression was low and only a minority of tran-
scripts (357) was changed more than 2-fold. Surprisingly, IVF
embryos were different from IVC blastocysts cultured in WM 20%
O2(3058 transcripts were statistically different but only 98 transcripts
were changed more than 2-fold; Giritharan et al., 2007). Fernandez-
Gonzalez et al. (2009) compared the gene expressions of IVC
embryos cultured in KSOM versus KSOM + fetal calf serum (FCS),
and observed that the presence of FCS during IVC affected several
genes involved in regulating epigenetic mechanisms. Their results are
particularly relevant because the same authors found that adult
animals resulting from IVC embryos cultured in KSOM + FCS dis-
played alteration of post-natal development and behavior (Fernandez-
Gonzalez et al., 2004).
The above-mentioned studies and the present findings support the
conclusion that each manipulation and culture conditions have an
independent effect on the transcriptome of the developing embryo.
Interestingly, the ICSI manipulation results in a higher number of
genes being changed compared with all the other manipulations
It is possible that the different pattern of calcium oscillation follow-
ing ICSI (Kurokawa and Fissore, 2003) triggers a different wave of gene
activation and therefore the effect of different culture media is less
prominent. Consistent with this possibility is the finding that the
S100a6 (S100 calcium binding protein A6), a calcium binding
protein, is down-regulated more than 25-folds in the ICSI groups.
S100a6 induces conformational changes and post-translational modifi-
cations of multiple cytoplasmic proteins (Santamaria-Kisiel et al.,
2006). In particular, it interacts with p53 and modulates apoptosis
(Grigorian et al., 2001).
Implication for future health
The most important clinical question relates to the long-term signifi-
cance of the observed gene changes. These changes could be self-
limited and isolated, like T-wave changes found on a healthy person
EKG following intense physical exercise. In fact, while one rodent
study found differences in selected gene expression in blastocysts gen-
erated by ICSI with mature spermatozoa or round spermatid injection
(ROSI; Hayashi et al., 2003), other studies did not find any obvious
long-term health consequences in ROSI mice (Tamashiro et al.
1999; Meng et al., 2002). On the other hand, it is possible that
some of the differentially regulated genes might alter the development
of additional genes, and as a result, potentially affect cellular and tissue
development, as asserted by the developmental origin of health and
disease hypothesis (Barker, 1998). Indeed, a cohort of ART pubertal
children has been found to have slightly worse metabolic parameters
than spontaneously conceived children of infertile couples (Ceelen
et al., 2008). We believe that our database provides fundamental
resources for understanding how the method of fertilization and
culture conditions affect mouse preimplantation embryo develop-
ment. Therefore the provided gene list could be useful to uncover
the cellularmechanisms that can
Overall, it appears that ICSI, IVF and in vivo produced mouse blas-
tocysts have a very different transcriptome.
lead to long-termhealth
Supplementary data are available at http://humrep.oxfordjournals.
G.G.: Conception and design, collection and assembly of data and
manuscript writing. M.W.L.: Collection of data. F.S.: Collection of
data. F.J.E.: Data analysis and interpretation. J.A.H., A.D., E.M.: Data
analysis and interpretation. K.C.K.L.: Collection of data. P.R.: Con-
ception and design, manuscript writing and final approval of
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