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
generated mouse embryos. This provides an overview of the transcriptional 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
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 ICSIWM
and 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 fertilization 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. ). ICSI embryos have an increased dysregulation (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 differentiation (Fig. 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 ): mitochondrial dysfunction and a disproportionately higher number of metabolic pathways 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
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 mechanism 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 γ amino butyric (GABA) receptor signaling and invasiveness signaling are prominent. Autocrine and paracrine GABA signaling via GABAA
receptors slow preimplantation embryonic growth by decreasing proliferation 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 chromosome, 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 placenta function is compromised. Among the transporter genes (Table ), 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
(solute carrier family 39, member 4) encodes for a transmembrane protein required for zinc uptake in the intestine. Mutations in this gene result in acrodermatitis enteropathica, a rare inherited defect in the absorption of dietary zinc (Wang et al., 2002
(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 ). Among them Cd81
are changed more than 10-fold. Cd81
encodes a tetraspanin that functions as an organizer of multi-molecular membrane complexes and as a result, regulates cell migration, fusion and signaling (Hemler, 2005
is located close to the cluster of imprinted genes on murine chromosome 7 which is syntenic with the Beckwith–Wiedemann syndrome associated cluster of imprinted gene on human chromosome 11p15.5 region (Paulsen et al., 1998
). Lim et al.
) reported that children born after ICSI with 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 conclusions 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 placental 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 transcribed) 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 misregulated. Importantly Wbp7, Smarca1 and Hdac6 are altered in ICSI embryos.
belongs to the histone deacetylases (HDACs) family of enzymes that catalyze the removal of acetyl groups from lysine residues 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 premature chromatin compaction in mouse oocytes and embryos (Verdel et al., 2003
(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
(WW domain binding protein 7) is a histone methyltransferase 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
The transcriptome comparison of ICSI embryos cultured in KSOMaa or WM and the comparison of ICSIWM and IVFWM blastocysts provided 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 ICSIKSOMaa
, it appears that while ICSIWM
embryos 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; Slc27a2; Slc7a3).
Finally only two pathways were statistically different between ICSIKSOMaa and ICSIWM (RhoA signaling and one carbon folate pathways). Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion. Down-regulation of Mthfd2 (methylenetetrahydrofolate dehydrogenase—NADP+-dependent—2) is, however, 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 IVFWM and ICSIWM confirms 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 IVFWM and ICSIWM (984 gene) as between ICSIKSOMaa and in vivo (1016) or ICSIWM and in vivo (947).
Pathways analysis confirms that similar pathways (Mitochondrial function pathways and metabolic pathways) are changed between IVFWM blastocysts and ICSIWM embryos 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 IVFWM
blastocysts (Table ). 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
(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 reticulum. Mutations in this gene have been associated with various forms of glycogen storage disease (Schaub and Heyne, 1983
Three epigenetic genes (Wbp7
) were differentially regulated in IVFWM
was down-regulated in ICSI embryos while Wbp7
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
also plays a key role in embryo development, and regulates the apoptosis and differentiation 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 analysis 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 synergistic increase in gene misregulation. IVC of zygotes in atmospheric oxygen (20% O2
) was associated with markedly increasing misregulated genes (20% O2
:WM: 354 genes changed more than 2-fold compared 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 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 transcripts (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.
) 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 displayed 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 analyzed.
It is possible that the different pattern of calcium oscillation following 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 modifications 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 significance 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 generated 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 development. Therefore the provided gene list could be useful to uncover the cellular mechanisms that can lead to long-term health consequences.
Overall, it appears that ICSI, IVF and in vivo produced mouse blastocysts have a very different transcriptome.