comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and...

MOLECULAR REPRODUCTION AND DEVELOPMENT 74:512–519 (2007)

Comparison of Mice Born After IntracytoplasmicSperm Injection With In Vitro Fertilization andNatural MatingTREVOR J. WILSON,1 ORLY LACHAM-KAPLAN,2 JODEE GOULD,1 ANDREW HOLLOWAY,3

IVAN BERTONCELLO,3 PAUL J. HERTZOG,1 AND ALAN TROUNSON2*1Centre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Monash University,Clayton, Victoria, Australia2Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia3Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia

ABSTRACT The procedures of in vitro fertili-zation (IVF) and intracytoplasmic sperm injection(ICSI) are routinely used in modern medicine toovercome infertility and, in animal husbandry, topropagate lines with compromised fertility. However,there remains concern that manual selection andinjection of whole sperm into oocytes could contributeto pre- and postnatal developmental defects. Toaddress this, we have used gene expression profilingand immunophenotyping to characterize offspringgenerated by these procedures. We used gametes fromglutathione peroxidase 1 knockout (Gpx1�/�) mice asa sensitized screen responsive to oxidative stress fromartificial reproduction technologies (ART). There wereno differences between IVF and ICSI derived offspringin gene expression patterns, and minor differences inhematopoietic parameters. Furthermore there wereonly minor differences between these IVF and ICSIpups and those derived from natural mating. Thesedata demonstrate for the first time in that there is nosignificant phenotypic affects of ICSI when comparedto IVF and we identified a relatively minor influence ofthe artificial fertilization methods on phenotype ofoffspring compared with natural mating. These ob-servations would support the use of ICSI for derivationof mutant mouse lines and may be of some importancefor the use of this technique in human ART. Mol.Reprod. Dev. 74: 512–519, 2007.� 2006 Wiley-Liss, Inc.

Key Words: IVF; ICSI; oxidative stress; mouse

INTRODUCTION

Intracytoplasmic sperm injection (ICSI) (Palermoet al., 1992) has been developed as a routine andaccepted method for achieving fertilization. The advan-tage of ICSI is that it bypasses all the primary barriers tosperm-oocyte fusion associated with conventional IVF,including the zona pellucida, which only allows hyper-motile acrosome reacted sperm to penetrate the oocyteinvestments and fuse with the oolemma (Yanagimachi,

1994). ICSI has become a popular and accepted methodof fertilization in vitro in cases of human male infertility(Palermo et al., 1992). In addition, it is proving to be avaluable tool in animal husbandry, especially in experi-mental mouse genetic programs such as ENU (ethylni-trosourea) and other genome-wide mutagenesisinitiatives (e.g., Mutant Mouse Regional ResourceCentre Program). In these programs large numbers ofmouse lines need to be cryopreserved and ICSI is animportant complement to sperm cryopreservation, sinceit overcomes the necessity for recovery of highly motilesperm for fertilization. ICSI is also a useful technique formaintaining transgenic lines where reduced malefertility is a serious obstacle to fertilization in vivo orin vitro.

Concerns have been raised that the manual selectionof a spermatozoon and injection of the whole cell,complete with all acrosome and plasma membranesintact, results in abnormalities of sperm decondensa-tion, retention of the acrosomal structures and proteins,and exclusion of the paternal nuclear centriole from themitotic apparatus (Hewitson et al., 1999; Terada et al.,2000). These concerns have led to predictions ofchromosomal abnormalities and developmental defectsthat could be transmitted to offspring conceived by ICSI(Schatten et al., 1998). It has also been associated withepigenetic errors of genomic imprinting because of theapparent over-representation of children conceived byICSI with Prader–Willi syndrome and Angelman

� 2006 WILEY-LISS, INC.

This article contains supplementary materials available via Internetat http://www.interscience.wiley.com/jpages/1040-452X/suppmat/

Grant sponsor: National Institute of Child Health and HumanDevelopment; Grant sponsor: National Center for Research Re-sources; Grant number: U01 HD38228.

*Correspondence to: Prof. Alan Trounson, Monash Immunology andStem Cell Laboratories, Level 3, STRIP1, Monash University, Well-ington Road, Clayton Victoria 3800, Australia.E-mail: [email protected]

Received 28 July 2006; Accepted 14 August 2006Published online 22 September 2006 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20644

syndrome due to hypomethylation of the SNRPN (smallnuclear ribonucleoprotein polypeptide N) maternalallele (Cox et al., 2002; Orstavik et al., 2003). Maheret al. (2003) reported an over-representation of childrenborn after ART (6/149 or 4%) with Beckwith–Weide-mann syndrome (an epi-mutation on chromosome11p15.5) while only �1.2% of children are born afterartificial reproduction technologies (ART) in the generalpopulation. Interestingly, the six children from ARTwith the disorder included both IVF and ICSI (threecases each). A study by Halliday et al. 2004, also showedthat the risk of Beckwith–Weidemann syndrome inliveborn babies from IVF was 1/4,000, nine times greaterthan in the naturally conceived population. However,again this study did not distinguish between offspringproduced by conventional IVF and ICSI. Furthermore,methylation abnormalities of the 15q11-q13 region werenot detected in children born after ICSI (Manning et al.,2000). It would therefore seem likely that epigeneticimprinting disorders are not ICSI specific but areobserved also in IVF offspring. These disorders may bedue to in vitro manipulation and/or in vitro cultureeffects on events such as methylation, which are knownto influence the expression of imprinted genes (Dohertyet al., 2000; Khosla et al., 2001). IVF and in vitro culturehave also been reported to alter early embryonic geneexpression patterns (Stojanov and O’Neill, 2001).

To further investigate the potential effects of ICSI onphenotype of offspring which would include any majoralteration to gene expression and hematopoiesis, wehave used the mouse model system that has been usedextensively for IVF and ICSI quality control proceduresand is amenable to phenotypic and genotypic analyses.Furthermore, we have utilized the glutathione perox-idase 1 (Gpx1�/�) knock-out mouse line as a sensitizedscreen for abnormalities that might occur as a result ofICSI.Gpx1 is an enzyme involved in the cellular removalof superoxide which is generated during normal oxida-tive metabolism. Consequently, cells from the Gpx1�/�mouse are susceptible to oxidative stress (de Haan et al.,1998, 2004; Crack et al., 2001, 2003) and would thereforebe extremely sensitive to stresses that might be expectedto occur during micromanipulation and culture in vitroduring ICSI and IVF procedures. Gpx1�/� mice wereproduced by natural mating, IVF and ICSI, andanalyzed for alterations in general well being, inhematopoiesis (by immunophenotyping) and by globalgene expression profiling (using microarrays of �15,000murine cDNA sequences). We selected these analyticmethods because any chromosomal abnormality, epige-netic modification, or developmental abnormality wouldresult in the alteration in the expression of many genesand major changes in immunological parameters.Mouse embryo development and molecular geneticsare most commonly used to assess new ART methods,and for maintaining quality control of in vitro techni-ques in human IVF, but there is relatively littleinformation on the developmental and epigenetic nor-mality of conception by ICSI in the mouse. We con-sidered that data on mouse development and phenotype

were an important aspect of safety for the ICSIprocedure which bypasses most of the natural barriersfor fertilization.

MATERIALS AND METHODS

Assisted Reproduction Procedures

The Gpx1�/� knockout mice used in this study werebred on a mixed genetic background (129SV�C57BL6)(de Haan et al., 1998). Females of 6–8 weeks of age to beused as oocyte donors were superovulated by injection(SC) of 10 IU of equine chorionic gonadotropin (eCG,Folligon; Intervet, Lane Cove, Australia) and 10 IUhuman Chorionic Gonadotrophin (hCG; Choralon;Intervet) 50 hr later. Mature metaphase II oocytes wererecovered from the oviducts of superovulated females13–14 hr after hCG injection. Oocyte-cumulus cellcomplexes were liberated into warm HEPES-KSOMmedium (Biggers et al., 2000) containing 40 IU bovinehyaluronidase (Fraction IV-S; Sigma Chemicals Co. St.Louis, MO) for 3–5 min, to separate cumulus cells fromthe oocytes. Cumulus-free oocytes were collected andwashed in fresh warm HEPES-KSOM before culture inKSOM-AA medium (Biggers et al., 2000), The mediumwas equilibrated at 378C in a 5% CO2 in air overnightbefore use. Oocytes were maintained in the pre-equilibrated medium under mineral oil (Sigma) at378C in 5% CO2 in air for 1.5 hr until used formicroinjection and IVF.

Sperm for IVF and ICSI were obtained from 10 to14 weeks old males by removing one cauda epididymisfrom each of two males. A single slit was made in thetissue before it was placed in 2 ml warm and equilibratedmodified Tyrode’s medium (MT6) (Fraser, 1984) in a35 mm culture dish covered by mineral oil (Sigma) toallow sperm to swim out into the medium. Sperm werecapacitated for 2–3 hr at 5% CO2 in air at 378C beforeuse for insemination or microinjection (Fraser, 1984).

For IVF, 10 cumulus-denuded oocytes were trans-ferred to the capacitated sperm solution for 3 hr, thenwashed twice and cultured in KSOM-AA culturemedium under oil in an atmosphere of 5% CO2 in air at378C.

For ICSI, oocytes were placed in 40 ml droplets ofHEPES-KSOM medium in an oil filled 150 mm tissueculture dish (Falcon, Becton Dickinson Labware, Lin-coln Park, NJ). Sperm were mixed (1:1) with 20%polyvinylpyrolidone (PVP; Sigma) in HEPES-KSOM. A10 ml drop of the sperm-PVP mixture was placed in thetissue culture dish. Sperm were randomly selected andaspirated into a glass microinjection pipette with aninner diameter of 5 mm mounted on a Leica manuallyoperated micromanipulator (Leica Microscopy SystemsLtd., Wetzlar, Germany) attached to a Piezo system(Burleigh, USA). Micromanipulation was performed atroom temperature. Sperm heads were separated fromtails using a high Piezo pulse before being collected intothe injection pipette. Five sperm heads were aspiratedinto the injection pipette. Only then were five oocytesplaced in one of the HEPES-KSOM 40 ml drops. The

Molecular Reproduction and Development. DOI 10.1002/mrd

ANALYSIS OF ICSI, IVF, AND NORMALLY DERIVED PROGENY 513

injection pipette was moved into the drop containing theoocytes. A Piezo pulse was applied and a hole wascreated in the zona pellucida. The injection pipette wasinserted into the perivitelline space and a sperm headwas brought as close as possible to the opening of thepipette. The oolemma was ruptured by applying a gentlePiezo pulse and the isolated sperm head was injecteddeep into the ooplasm.

Following injection or insemination, oocytes weretransferred to equilibrated drops of KSOM-AA culturemedium. After 24 hr, 2-cell cleavage stage embryos weretransferred to pseudopregnant recipient (C57BL6�CBA) females mated to vasectomized males. Recipientswere allowed to give birth and the pups were weaned4 weeks later. Microarray and immunophenotypeanalyses were undertaken at 6–8 weeks of age.

Immunophenotyping

Groups of at least three age and sex matched mice(females) 6–8 weeks of age generated from naturalmating, ICSI or IVF were analyzed for a broad spectrumof hematopoietic and immunological parameters aspreviously described (Bertoncello and Williams, 2001).Briefly, blood was collected into sodium citrate contain-ing tubes and thymus, spleen and femurs were removedinto sterile PBS containing 2% fetal bovine serum. Cellsuspensions prepared by gently extruding throughstainless steel sieves (thymus, spleen) or flushing witha needle and syringe (femur). Total cell counts from eachorgan were determined using an automated counter andsamples were stained with a range of cell markers forFACS analysis. Platelet, WBC and RBC counts andHematocrit percentage were also determined fromperipheral blood, before RBC lysis and similar FACSanalysis. Markers used were CD3, CD4, CD8, B220,IgM, IgD, Mac1, and Gr1. Numbers of progenitor cells inbone marrow and spleen were quantitated using culturesystems which determine the number of high and lowproliferative colony forming cells (Bertoncello andWilliams, 2001). All data are shown as mean�SD andwere statistically analyzed using the two-tailed t-test.

cDNA Microarray analysis

Since the brain ofGpx1�/�mice brain has been foundto be very sensitive to injuries and oxidative and otherstresses (de Haan et al., 1998, 2004; Crack et al., 2001,2003), this organ was chosen for detailed gene expres-sion profiling analyses of the effects of ICSI and IVF onthe offspring. Briefly, brains were removed from threeage and sex matched progeny (males) from eachtreatment group and immediately snap frozen in liquidnitrogen. Total RNA was prepared using an ‘‘RNeasyKit’’ according to the manufacturer’s instructions (Qia-gen). Twenty-five micrograms of RNA from threeanimals were pooled and labeled RNA prepared byintegration of Cy3 dCTP or Cy5 dCTP nucleotides asdescribed (Hegde et al., 2000). The resultant labeledRNA, from samples to be compared, were hybridizedin pairs (Cy3 or Cy5 labeled) onto slides containing the15K NIA mouse cDNA cloneset printed at the Peter

MacCallum Cancer Institute Microarray Facility(Tanaka et al., 2000). Slides were scanned on a PackardScanArray 5000 (Perkin Elmer) and data analyzedusing Genespring software. Each sample was labeledand compared twice (using alternate dyes) with twoseparate treatment groups (i.e., ICSI vs. IVF, IVF vs.Natural matings and ICSI vs. Natural matings). Micro-array data were normalized using the Lowess algorithmand all data shown is from combined dye swap experi-ments.

Semi-Quantitative RT-PCR

One microgram of Total RNA (prepared as above fromthree male progeny from each fertilization procedure)was used in reverse transcription (RT) reactions usingoligo-dT primers to prepare 1st strand cDNA. Onehundred nanograms of these samples, together withnon-RT treated controls, were then used in PCRreactions for specific genes. Egr1 was detected usingthe oligonucleotides 50-TATCCCATGGGCAATAGAGC-30 and 50-AGGCTTTGAGAGCAAAAACG-30. Arc wasdetected using the oligonucleotides 50-CCTATGC-CAGCCAAATGAGT-30 and 50-CTTGTGCTGGTAGCTGCTTG-30. Gapdh control was detected using oligonu-cleotides 50-CTGCCACCCAGAAGACTGTGG-30 and 50-GTCATACCAGGAAATCAGC-30. Twenty-five and thirtycycles of amplification were used to ensure that ampli-fication was in the linear range.

Ethics

Monash University Animal Ethics Committeeapproved (approval number 38/1999) all experimentsin accordance with the NH and MRC guidelines.

RESULTS

Gene Expression Profiling of ICSI andIVF-Derived Offspring

Since previous studies had demonstrated only minorchanges in gene expression in the brains of Gpx1�/�mice compared to Gpx1þ/þ controls and this organ wasparticularly sensitive to stress, we compared the globalgene expression in the brains of offspring derived fromICSI and IVF. This expression analysis was performedusing the Gpx1�/� offspring as a sensitized screen todetect any genetic changes. When gene expression in thebrain of IVF or ICSI conceived Gpx1�/� offspring werecompared with offspring of naturally mated Gpx1�/�mice, of the �15,000 genes examined the expression of21 (0.0014%) sequences, comprising 12 defined genesand 9 unidentified ESTs (expressed sequence tags) were>two-fold different in expression; the expression levelsof>99.9% of genes were within the limits set as ‘‘normal’’in this experiment (Fig. 1A,B). The details of these21 genes and relative expression levels are shown inTable 1. Of these alterations, the expression of 19/21genes were increased in the in vitro fertilized offspringrelative to controls. The degree of altered gene ex-pression ranged from two- to five-fold. Cerebellin 1precursor protein, known to be enriched in Purkinje cellpost-synaptic structures (Urade et al., 1991), was


514 T.J. WILSON ET AL.

increased by almost five-fold. Other genes such as E2Ftranscription factor 6 and chromobox homolog 5, bothinvolved in chromatin binding (Ogawa et al., 2002;Cheutin et al., 2003), were increased more than two-foldin both IVF and ICSI derived animals. The only twogenes which showed decreased expression were Egr1and Arc. These genes encode a hypoxia inducedtranscription factor and a protein believed to be involvedin neuronal long-term potentiation respectively(Guzowski et al., 2000; Nishi et al., 2002), Semi-quantitative RT-PCR was used to confirm the decreasedexpression. However, these changes, although signifi-cant, were less than two-fold (Fig. 2 and data not shown).Egr1 was decreased by 1.4-fold in IVF and ICSI derivedanimals whereas Arc was decreased 1.2-fold in IVF and1.1 fold in ICSI derived animals. It was notable that thesame genes were altered in IVF and ICSI derived mice.Indeed, when the microarray experiment was per-formed by a direct comparison between the ICSI andIVF derived mouse brains, there were no genes whoseexpression was greater than the two-fold limit ofdifference set for this experiment.

Immunophenotype of ICSI andIVF-Derived Offspring

Another system that we have found to be exquisitelysensitive to environmental changes and stresses(Bertoncello and Williams, 2001) is the hemopoieticsystem, the components of which can be measured inblood as well as organs of hemopoietic development suchas the bone marrow, spleen, and thymus. A number ofparameters were investigated including blood composi-tion, cell numbers of lymphoid organs, relative levels ofT- and B-lymphocyte subpopulations, and numbers ofmacrophages and granulocytes (in total 17 differentmolecular phenotypes in 4 organs¼ 68 parameters). Forthe majority of these parameters no significant differ-ences were observed (Table 2 and supplementaryTable 1). Furthermore, these animals remained healthyin a conventional animal facility. However, a fewdifferences were observed between ICSI and IVF off-spring of Gpx1�/� mice. These included the totalnumber of bone marrow cells in ICSI generated mice(23.4�1.8�106 cells/femur) compared with IVF gener-ated offspring (14.9� 1.4�106 cells/femur) which weresimilar to those from natural mating offspring(14.3�3.3�106 cells/femur). This increase in ICSI micemay be related to an expansion of progenitor cells whichis supported by observations of a reduced percentageof mature T-cells and an increased number of low-proliferative potential colony forming cells in their bonemarrow. In the thymus, there was a reduced number ofmature CD4-CD8þT-cells in animals generated by ICSIcompared with naturally generated animals (4.4�0.2%and 5.4�0.1%, P< 0.05). There was a similar but notsignificant trend in animals generated by IVF (3.6�1.1;P¼0.11). In the spleen there was a significant decreasein the percentage of both macrophages and granulocytes(Mac1 and Gr1 positive cells, respectively) in IVFgenerated progeny compared with controls whereas


Fig. 1. Comparison of microarrays of Gpx1 �/� mouse brains fromanimals generated by natural Matings or IVF (A), Natural matings orICSI (B) and IVF or ICSI (C). Genes expressed at equal levels betweensamples fall along the midline of the plot, with spots outside the linesindicating genes with 2 fold changes between samples.


only the granulocyte decrease was significant in animalsgenerated by ICSI (P<0.05). This was accompanied byan increased percentage of immature B lineage cells inthe spleen in both IVF and ICSI generated progeny.

DISCUSSION

In the present study we demonstrate that the in vitroembryo manipulation procedure of ICSI induces noadditional changes in the normality of offspring relativeto conventional IVF procedures as measured by thefollowing parameters in the 6-week-old mouse modelsystem:

* Gene expression profiling of brain samples of theoffspring

* Extensive immunophenotyping of the hemopoieticsystem of the offspring

* General health of the Gpx1�/� genetically mod-ified mouse line.

We chose the Gpx1 knockout mouse as a ‘‘sensitizedscreen’’ for possible abnormalities induced by IVF orICSI. We have previously shown that these micedemonstrate normal fertility and reproduction andappear essentially normal, however cells from thesemice are sensitive to oxidative stress (de Haan et al.,1998, 2004). Therefore, we reasoned that gametes fromthese mice would be more sensitive to culture conditions,in vitro manipulation and fertilization procedures. Inaddition, our extensive studies have demonstrated thatthe brains of Gpx1�/� mice are particularly sensitive todamage including aging, ischemia-induced infarction,

and inflammation and cell death associated withneurotrauma (Crack et al., 2001). Hence, it is aremarkable result that very few abnormalities wereobserved in the offspring derived from IVF and ICSIwhen using gametes from these sensitive mice.

Expression profiling using gene microarrays enablesan exquisitely sensitive global snapshot of the molecularstatus of a cell. Using the NIA murine cloneset of�15,000 genes, we demonstrated that when comparingthe brains of mice derived by natural matings to eitherthe IVF or ICSI technique, almost all genes (>99.9%)were expressed at levels that are within the two-foldlimits set on the experiment. Notably, there was nodifference, at this level of stringency, between geneexpression in the brain of mice derived by either ICSI orIVF. The 21 genes that changed expression wereprobably the result of in vitro manipulation and/orculture. Importantly, two representative gene expres-sion changes were validated by another procedure,semi-quantitative RT-PCR. Only one of the genes waschanged as much as five-fold, the cerebellum 1 precursorprotein, which suggests that it should be furtherinvestigated for potential consequences in the offspringof ART. Interestingly, two genes, E2F transcriptionfactor 6 and chromobox homolog 5, which have beensuggested to be involved in chromatin binding andremodeling (Ogawa et al., 2002; Cheutin et al., 2003),were increased in both IVF and ICSI generated animals.Not only was there no additional effect of the ICSIfertilization procedure on global gene expression inbrains of 6-week-old offspring, but their susceptibilityto mid-cerebral artery occlusion (MCAO)-induced cere-bral ischemia was also similar (data not shown).


TABLE 1. Genes Demonstrating Altered Expression in Gpx1(�/�) Mice Derived From Naturally Mating or ART*

Geneidentifier Name

Relativeexpression–IVF

Relativeexpression—ICSI

BG070825 Early growth response gene 1 0.28 0.4BG067360 Activity regulated cytoskeletal-associated protein 0.48 0.48BG070102 Putative homeodomain transcription factor 2.4 2.1BG072629 Ewing sarcoma homolog 2.8 2.1BI076713 EST from newborn ovary cDNA library 2.0 2.1C75991 Chromobox homolog 5 (Drosophila HP1a) 2.0 2.2AW546550 EST from F mesonephros/gonad cDNA library 2.8 2.2BG073080 EST from pooled cDNA library 2.5 2.2AW550998 EST from F mesonephros/gonad cDNA library 2.4 2.3AW548440 Ribosomal protein S15 2.6 2.3AW549180 EST, Weakly similar to A45964 hemoglobin alpha chain—mouse

[M. musculus] 48% identity2.9 2.3

AW551388 E2F transcription factor 6 2.8 2.3C79876 EST blastocyst cDNA library 2.6 2.4BG063119 ESTs, weakly similar to glycoprotein [R. norvegicus], expressed

neurons, liver, retina, spleen, lung tumours2.7 2.3

AW550525 EST from F mesonephros/gonad cDNA library 2.8 2.4BG072975 EST from pooled cDNA library 2.6 2.4BG073492 DNA segment, human D4S114 neuronal protein NP31

(widely expressed)2.0 2.8

BG070209 Cerebellin 1 precursor protein 3.2 4.2BG072807 Carbonic anhydrase-like sequence 1 1.8 5.3BG073469 Hemoglobin, beta adult major chain 2.6 2.4

*Analysis of gene expression in brains from progeny derived from natural mating, IVF or ICSI. Expression of genes in ARTderived animals are shown relative to naturally born animals.


Furthermore, the observation of these similarities in thesensitized mouse Gpx1�/� line emphasizes the conclu-sion that ICSI induces no different effects in derivedoffspring to IVF.

The other extensive analysis was immunophenotyp-ing of hemopoiesis, since it is well established that thissystem is sensitive to the general environment, stress,and disease. Subtle immune alterations were observedin animals from IVF and ICSI generated embryosconsistent with increased proportions of immatureimmune cells, which could increase the predispositionto infection. However, all the animals remained healthywhen housed in a conventional animal house and anyimpact of these changes may only be observed if IVF/ICSI mice were exposed to infection or other immunechallenge. Consistent with the above conclusions, thegreatest changes occurred to a similar degree in micederived by both ICSI and IVF and only subtle differencesin a few parameters were observed between the twofertilization procedures.

In addition to providing direct evidence of the minimalchanges that occur in the offspring of ART embryos,whetherbyICSI orconventional IVF, our study has signi-ficant implications for the effects of these procedures


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in human assisted reproduction. Consistent with ourstudies, a detailed analysis of embryonic chromosomesshowed no difference between in vitro fertilized humanembryos (61%) and ICSI embryos (52%), nor was thereany difference in gonosomal aneuploidies (Munne et al.,1998). Furthermore, there was no increase in theincidence of chromosomal abnormalities reported inspontaneous miscarriage between IVF and ICSI con-ceived human embryos (Causio et al., 2002). Monosomyof the X chromosome (45, XO) was the most frequentabnormality observed. Examination of 1586 prenatalkaryotypes of ICSI conceptuses showed an increase ofchromosomal abnormalities of 1.6%, compared to 0.5%(natural conception of maternal age of 33.5 years), duemainly to sex chromosomal abnormalities (Bonduelleet al., 2002b). Consistent with these studies, a large-scale follow-up of ICSI children showed no increasedrisk of major congenital malformations when comparedto IVF children (Bonduelle et al., 2002a, 2003). However,Swedish studies showed an increase in major birthdefects in children born after ICSI but these defectsmight be accounted for by adjusting for multiple births(Wennerholm et al., 2000).

In contrast, several studies have reported birthdefects in ICSI and IVF offspring. Kurinczuk and Bower(1997) reported an excess of major cardiovasculardefects, genitourinary defects and gastrointestinaldefects in ICSI conceived children in Western Australia,and Hansen et al. (2002) reported increased major birthdefects in both ICSI and IVF children at 1 year of age inWestern Australian birth registries. Another analysis ofa large cohort of ICSI children from Germany found anincreased risk of major malformations when comparedto the whole of population-based birth registry (Ludwigand Katalinic, 2002). A mild delay in development ofICSI children was observed at 1 year of age by Bowenet al., when compared to IVF or naturally conceivedchildren. It would be expected that these malformationsand developmental delay would coincide with altera-tions in the expression of a large number of genes.However, in the current studies we observed noalterations in gene expression that would be consistentwith developmental and epigenetic abnormalitiesreported in human ART. It should be noted however,that human embryos are normally grown to the 2- to 8-cell stage in vitro for 2–3 days after insemination andICSI before transfer to the uterus. In some clinicsin vitro culture to the blastocyst stage may take 5–6 days. In the present experiments the mouse embryoswere grown for only 1 day to the 2-cell stage beforetransfer in order to minimize the potential stress effectsof in vitro culture on the sensitive Gpx1�/� strain. Wehad already noted the difficulty in growing the 129/BL6wild-type embryos to the blastocyst stage. To adequatelycompare the IVF and ICSI methods, the possible in vitroculture effects were minimized and thus we cannotexclude effects or interactions due to increased culturetime in vitro.

We conclude that ICSI does not significantly changethe phenotype of offspring when compared with IVF.

Both ICSI and IVF are associated with minor changes ingene expression in the brain and also minor hemato-poietic parameters in offspring. These data support theuse of ICSI as an alternative to IVF for derivation ofmouse lines in large-scale breeding and mutagenesisprograms.

ACKNOWLEDGMENTS

This study was conducted as part of the NationalCooperative Program on Mouse Sperm Cryopreserva-tion sponsored by the National Institute of Child Healthand Human Development and the National Center forResearch Resources (U01 HD38228). The authors thankBrenda Williams for assistance with Flow cytometryand Monika Generowicz and staff for assistance withanimal husbandry.

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comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and...

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