Thierry Voet

Evelyne Vanneste

Niels Van Der Aa

Cindy Melotte

Sigrun Jackmaert

Tamara Vandendael

Matthias Declercq

Sophie Debrock

Jean-Pierre Fryns

Yves Moreau

Thomas D'hooghe

Joris R Vermeesch
email: joris.vermeesch@med.kuleuven.be

Breakage-fusion-bridge cycles leading to inv dup del occur in human cleavage stage embryos

Keywords: Breakage-fusion-bridge (BFB) cycle, inv dup del, cleavage stage embryogenesis, single-cell microarray analysis, chromosome instability (CIN), in vitro fertilization Breakage-fusion-bridge (BFB) cycle, inv dup del, cleavage stage embryogenesis, single-cell microarray analysis, chromosome instability (CIN), in vitro fertilization

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Introduction

Recently, it was shown that chromosomal rearrangements occur frequently in the human cleavage stage embryo by microarray DNA-copy number profiling of all blastomeres separately from multiple embryos following in vitro fertilization (IVF) [Vanneste et al., 2009a]. Seventy percent of the top quality IVF embryos derived from young and fertile partners were mosaic for megabase-sized segmental aneuploidies that were characterized by complementary deletions and duplications or amplifications in different blastomeres of a single embryo. Frequently the embryo also carried blastomeres with a normal copy number of the chromosome that was rearranged in the sister blastomeres [Vanneste et al., 2009a]. Other studies that used microarray technology or metaphase comparative genomic hybridization (CGH) to type DNA copy number in single blastomeres revealed the occurrence of segmental aneuploidies in less than 38.5% of the embryos [Voullaire et al., 2000;Wells and Delhanty, 2000;[START_REF] Voullaire | Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure[END_REF]Daphnis et al., 2008;[START_REF] Hellani | Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening[END_REF]Johnson et al., 2010;[START_REF] Treff | Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays[END_REF]. Based on the patterns of the DNA imbalances that were observed by single-cell array CGH, a number of predictions were made about the events leading to those imbalances. First, DNA double-stranded breaks (DSBs) would occur in the zygote or the first two cleavage cell cycles. The cause of these breaks remains unknown. Since approximately 36% of sperm nuclei derived from normospermic men contained DSBs which increased to ~57% in oligospermic men [Derijck et al., 2007] and parent-of-origin studies on postnatal cases of non-recurrent terminal deletion chromosomes demonstrated a preponderant loss of the paternal allele [Overhauser et al., 1990;Dallapiccola et al., 1993;Cody et al., 1997;Crow, 2000] we hypothesized that the postzygotic structural chromosome instability might be triggered by the DNA-breaks present in ejaculated human spermatozoa. If so, the Deleted: [Vanneste et al., 2009] Deleted: [Vanneste et al., 2009] Deleted: 5% to Deleted: [Daphnis et al., 2008;[START_REF] Hellani | Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening[END_REF]Johnson et al., 2010;Voullaire et al., 2000;[START_REF] Voullaire | Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure[END_REF]Wells and Delhanty, 2000] Deleted: [Cody et al., 1997;Crow, 2000;Dallapiccola et al., 1993;Overhauser et al., 1990]

Collection of embryos

For parent-of-origin analysis 24 embryos were used. Sixty-two blastomeres were biopsied from 11 PGD embryos following microdeletion screening or sex selection (all remaining blastomeres/embryo) [Vanneste et al., 2009a]. Embryos 6,8,9,10,14,15,17,18,19,20 and 21 of this previous study were included because at least 2/3 of the blastomeres derived from one embryo were informative for copy number analysis and DNA from both parents was available.

Twenty-one blastomeres were biopsied from 11 embryos for array-based-PGD for a complex chromosomal rearrangement including an interchromosomal insertion and reciprocal translocation in the male partner (maximum 2 blastomeres per embryo). These embryos were numbered 24 till 34. Ten blastomeres were biopsied from 2 embryos following FISH-based-PGD for a reciprocal translocation in the male partner (all remaining blastomeres/embryo). These embryos were numbered 35 and 36. All embryos were derived from young couples showing no indication for preimplantation genetic diagnosis for aneuploidy screening (PGS). Semen quality was determined for two separate samples with at least a 1-week interval and interpreted according to the World Health Organization (WHO) criteria [World Health Organization, 2010].

For microarray-guided FISH, one or two blastomeres were biopsied per embryo and analyzed by microarray analysis. Thirteen embryos with a chromosomal anomaly by microarray analysis as well as FISH results in the majority of the remaining nuclei are described in this study.

Numbering of these embryos is according to Supp. Table S1. Of these embryos, (1) 3 were 4 or 5-day-old in vitro fertilized (IVF) embryos which were genetically unsuitable for uterine transfer following PGD and (2) 10 were spare embryos, defined as 3-or 4-day-old IVF embryos of Field Code Changed Deleted: [Vanneste et al., 2009] Deleted: Thirty-nine embryos were used f Deleted: . Of each embryo, blastomeres of 11 embryos were analyzed on SNP array (1 blastomere per embryo, except for embryo 33 where two blastomeres were taken for array analysis), while the two other embryos were analyzed on BAC array (1 blastomere per embryo). The remaining cells were fixed for FISH analysis either simultaneously or after PGD.

Collection of single blastomeres from human embryos

Embryos were briefly incubated in acidic Tyrode's solution (pH 2.4) (Vitrolife, Gothenborg, Sweden) to remove the zona pellucida. They were then transferred to droplets of Ca 2+ -and Mg 2+free medium supplemented with bovine serum albumine (Sigma-Aldrich) and gently pipetted to disaggregate the individual blastomeres. For array analysis, a single blastomere with a clearly visible nucleus was washed three times in the dissociation medium and then transferred using a mouth-piece to a 200 µl PCR tube containing 1.5 µl alkaline lysis buffer (50 mM dithiothreitol (Sigma), 200 mM KOH). For FISH analysis, the blastomeres were washed twice with culture medium to get rid of possible oil remnants and were fixed on a Superfrost plus microscope slide (LaboNord) using 0.01 mol/l HCl with 0.1% Tween-20 (Merck), as described [Melotte et al., 2004]. Briefly, following lysis of the blastomeres, the nuclei were allowed to dry and became fixed on the glass slide. Finally, slides were washed in 1 X phosphate-buffered saline (PBS) for 5 min and dehydrated by sequential washing in 70%, 90% and 100% ethanol. Fixed nuclei were pre-treated for 20 min with 0.5 mg pepsin/ml (Sigma) in 0.01 mol/l HCl at 37°C followed by a 3 min wash in 1 X PBS. Post-fixation was performed by incubating the slides for 10 min in a 1% formaldehyde solution with 0.05 mol/l MgCl 2 and 1 X PBS at 4°C. Slides were subsequently washed in 1 X PBS and dehydrated by ethanol series.

Single cell array

A single blastomere was lysed and amplified following a multiple displacement amplification approach with Genomi Phi V2 (GE Healthcare) as described [Spits et al., 2006]. We did not use single cell amplifications yielding less than 2 µg DNA.

BAC arrays were performed in 36 hours as described [Vanneste et al., 2009a]. Briefly, 150 ng of single cell amplified DNA and non-amplified genomic reference DNA extracted from lymphoblasts of an individual with Klinefelter Syndrome were labeled for 2h by random primer labeling (BioPrime aCGH Genomic Labeling System; Invitrogen) using respectively Cy5-and Cy3-dCTPs (GE Healthcare). We mixed 1.75 µg of labeled single cell and reference DNA in hybridization solution and hybridized it overnight. Following scanning, we included only spots with signal intensities at least 2-fold above the autosomal median background intensity in the analysis which was performed using R (version 2.6. 1, R development Core Team, 2007).

GeneChip Human Mapping 250K NspI arrays (Affymetrix) for single blastomeres were performed in four days according to manufacturer's instructions using 250 ng of single cell amplified DNA. SNP-copy number analysis was performed to a publicly available reference pool of 41 HapMap females using either 'Copy Number Analyzer for Genechip (CNAG) version 2.0' [START_REF] Nannya | A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays[END_REF] or the 'Copy Number Analysis Tool' (CNAT4.0.1, Affymetrix). CNAT4.0.1 parameters were set as described [Vanneste et al., 2009a].

For SNP-typing, we analyzed probe intensities by the 'GeneChip Genotyping Analysis Software (GTYPE) version 4.1 (Affymetrix)' using the dynamic model [Di et al., 2005] with stringency P=0.12. LOH was performed using 'Copy Number Analyzer for Genechip (CNAG) version 2.0' [START_REF] Nannya | A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays[END_REF]. Deleted: [Vanneste et al., 2009] Deleted: as described [Vanneste et al., 2009] Deleted: . Briefly, we applied quantile normalization on the probe intensities and compared them by the 'Copy Number Analysis Tool' (CNAT4.0.1, Affymetrix) to a publicly available reference pool of 41 HapMap females. P Deleted: [Vanneste et al., 2009] Deleted: as well as copy number analysis Spectrum orange and Spectrum green (Vysis, Abbott laboratories, IL) were used as fluorochromes. Two to five clones of the same locus were mixed to obtain strong fluorescent signals. The final probe mixture was prepared as described [Melotte et al., 2004]. Briefly, the combined probemix, to which Cot-1 DNA was added, was dried and dissolved in hybridization buffer. Then, 1 µl of the probe was applied to the slide, covered with a coverslip (10 mm diameter) and sealed with rubber cement. The nuclei and probes were denatured simultaneously on a hot plate at 75°C for 5 min. Hybridization was allowed to take place overnight in a humid chamber at 37°C. After hybridization, excess or non-specific bound probe was removed by subsequent washes in 0.4 X standard sodium citrate buffer (SSC)/0.3% Igepal CA-630 (Sigma Aldrich) (73°C for 2 min), 2 X SSC/0.1% CA-630 (Sigma Aldrich) (room temperature for 1 min) and 2 X SSC (room temperature for 1 min) followed by dehydration through ethanol series. After drying, the slides were mounted in Vectashield anti-fading medium (Vector Laboratories, Peterborough, UK) containing 2.5 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Boehringer Ingelheim GmbH, Germany). Nuclei were examined using an Axioplan 2 microscope (Zeiss NV, Zaventem). For each additional FISH round, the coverslip was removed using PBS. The nuclei were denatured in 0.06 X SSC for 7 minutes in a warm water bath at 75°C, followed by a dehydration using sequential washes in 70% (ice cold), 90% and 100% ethanol, respectively. The probemix was denatured for 5 minutes in a warm water bath at 75°C. The quality of the probe mixture was first tested on nuclei derived from mitogen-stimulated blood lymphocytes. In 100 nuclei, the number of signals for each of the probes was counted. Detailed probe mix information S1. FISH probes flanking a chromosomal breakpoint were determined based on their position in NCBI35.

Parent-of-origin analysis

SNP probe intensities were analyzed by GTYPE 4.1 (Affymetrix) using the dynamic model [Di et al., 2005] with stringency P=0.12. The parent-of-origin algorithm determines in single cell genotypes the allelic origin of (aberrant) loci by identifying and visualizing in a parent-specific manner SNPs which demonstrate a Mendelian error. The algorithm was developed in R (version 2.8.1 -http://www.r-project.org/) and is available upon request.

SNPs fulfilling the rules described in Table 1, were scored as either +1 or +0.5 (maternal) or -1 or -0.5 (paternal).

Subsequently, these scores are plotted genome-wide in pdf format per chromosome.

Rationale behind the algorithm: If for a particular SNP the maternal and paternal genotype are respectively 'AA' and 'BB', one expects that the genotype of the SNP in a blastomere from an embryo of those parents would be typed 'AB'. However, if that SNP in the blastomere is called 'AA', this could be due to allele drop out (ADO) of the paternal B-allele, a preferential amplification (PA) of the maternal A-allele, a true deletion of that locus on the paternal chromosome or a true amplification of that locus on the maternal chromosome. Similarly, if for a particular SNP the maternal and paternal genotype are respectively 'AB' and 'BB', while 'AA' is obtained in the single cell, this could indicate a ADO of the paternal B allele, PA of the maternal A allele, a true deletion of the paternal B allele or a true amplification of the maternal A allele. To discriminate between single cell whole-genome DNA amplification artifacts and true DNA copy number alterations only consecutive SNPs are scored. ADO and PA produce random parent-of- 10 origin artifacts (alternating positive and negative scores for consecutive SNPs), while true chromosomal aberrations stand out in consecutive SNPs and are assigned to either maternal or paternal origin. For nullisomic regions the algorithm will produce random noise (alternating positive and negative scores for consecutive SNPs), while the majority of SNPs within hemizygous deletions are collectively assigned to either bin of maternal or paternal origin. The parent-of-origin data was thus applied to interpret SNP-copy number profiles.

Accession numbers

Data discussed in this publication have been deposited in the US National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE11663.

Results

Parent-of-origin algorithm

To determine the parental origin of the rearrangements seen in the blastomeres, we genotyped both parents and the single blastomeres using Affymetrix 250K SNP arrays. Because single-cell DNA needs amplification for genome-wide SNP typing, errors are introduced in the single-cell genotyping due to allele drop out or due to biases in DNA amplification. Hence, to enable parentof-origin analyses, we developed a novel algorithm that determines the allelic origin of (aberrant) loci by identifying and visualizing SNPs with a Mendelian error in a parent-specific manner at the genome wide level. To determine the accuracy of this approach, we typed a set of single blastomeres with known aneuploidies. For all hemizygous X-chromosomes in male embryos the maternal allele was correctly identified (26 blastomeres tested in 8 embryos). Parental origin were missing the maternal allele. In embryo 14 three blastomeres were lacking a maternal allele and one remaining blastomere was typed to lack both alleles and hence was nullisomic for chromosome 16. In embryo 18 all seven blastomeres carried an extra maternal allele explaining the trisomy 15. The accuracy of this approach to characterize segmental aberrations was further determined in ten blastomeres of two embryos derived from a couple carrying a paternal translocation t(7;21) (p11.2;q11.2). In both embryos that inherited a derivative chromosome 21 as well as a normal chromosome 7 by adjacent I segregation, the expected segmental 21q11.2-qter deletion of the paternal allele was correctly identified. SNP copy number analysis revealed the expected 7p11-pter duplication in seven blastomeres of which three could be confirmed by paternal origin. One blastomere carried two copies for the 7p11-pter segment and one copy of the 7p11-qter segment. Parent-of-origin typing revealed the loss of the normal paternal allele of chromosome 7 in this blastomere. In the two remaining blastomeres, the copy number profile for chromosome 7 was uninformative. Similar results were obtained for 21 blastomeres derived from embryos of another couple undergoing preimplantation genetic diagnosis (PGD) for a complex chromosomal rearrangement in the male partner. This demonstrates that the parent-of-origin algorithm accurately types the allele that is affected by chromosome missegregation or rearrangement. It also distinguishes homozygous from hemizygous deletions. Trisomic alleles could be determined with an accuracy of approximately 50%. In total, 93 blastomeres derived from 24 embryos were analyzed. Parent-of-origin typing allowed the characterization of mitotic non-disjunction events by identifying the allele that was lost in one blastomere but gained in a sister blastomere (Fig. 1).

Both paternal and maternal alleles were frequently rearranged

The analyses of all whole chromosome imbalances (n = 164) which were mosaic in an embryo or present in one or both blastomeres biopsied from embryos 24 to 34, are listed in Table 2. No significant difference was detected between the prevalence of mitotic loss of the paternal or maternal allele in potential mitotic-nondisjunction or anaphase-lagging events. Two meiotic monosomies and one meiotic trisomy, all maternal in origin, were detected in three different embryos respectively.

Segmental aneuploidies were considered simple if unaccompanied by another DNA imbalance for the same chromosome in the same blastomere. Seventeen simple events were detected in 11 of the 24 embryos (46%; 11/24) and comprised 33 segmental imbalances of telomeric loci. The identical parental origin of recurrent or reciprocal segmental aneuploidies in the sister blastomeres of an embryo indicated that these copy number changes resulted from one chromosomal break or rearrangement in a precursor blastomere. Eight events were paternal and five were maternal in origin. For seven duplications in five simple chromosome rearrangement events the parental origin could not be determined.

Thirty-nine complex rearrangements, characterized by either multiple aberrations for a single chromosome in the same blastomere or segmental uniparental isodisomies were observed in 9 of the 24 embryos (37,5%; 9/24). They are listed in Fig. 2 and described further below. Chromatids that lost a p-or q-terminal segment, while the remainder of the chromatid was duplicated or amplified in the same blastomere, were detected in 25% of the embryos (n=11; in 6 embryos; 25%; 6/24). Additional loss of the homologous allele in the same blastomere, resulted in 13 segmental uniparental isodisomy-null or amplification-null profiles (Fig. 2A). Of the potential isodicentric chromosomes, six were paternal in origin and five occurred on the maternal allele.

In general, no obvious correlation was apparent between post-zygotic chromosomal rearrangement and the semen quality of the couple (Supp. Table S2).

Terminal deletions flanking inverted duplications

Bipolar segregation of the isodicentric chromosomes can instigate breakage-fusion-bridge (BFB) cycles that are characterized by pure terminal deletions and terminal deletions accompanied by inverted duplications (Fig. 3A).

Parent-of-origin typing suggests the presence of inv dup del chromosomes in single cells (Fig. 3B). Chromosome 1 in blastomere "g" of embryo 20 had a p-terminal deletion which was immediately flanked by a Mb-sized duplication and subsequently a normal copy number profile for the remainder of this chromosome. If the deleted and duplicated segments of this chromosome were related as expected for inv dup del chromosomes, the segments should have the same parental origin. Both the deletion and accompanying duplication were found to be of maternal origin (Fig. 3B). Consistent with a maternal event, a reciprocal pure deletion of the maternal allele was detected in blastomere "c" (Fig. 3B). This strongly suggests that isodicentric formation and related BFB-cycles do occur in early human embryogenesis following IVF.

Whereas the parent-of-origin typing strongly suggests the existence of inv dup del chromosomes, interphase FISH would provide ultimate proof. To identify interphase blastomeres with a potential inv dup del rearrangement we applied microarray-guided FISH. In this approach, one or two blastomeres were biopsied and arrayed to search for a chromosomal anomaly that can be the complement of an inv dup del rearrangement in one of the remaining cells that were fixed 3C). In a second FISH-cycle with two differentially labeled probes distal from the breakpoint, the q-terminal duplication detected by microarray analysis was confirmed by the detection of an extra third FISH signal for both probes in a nucleus that produced two signals for each probe in the first FISH-cycle (Fig. 3C).

Signatures of ring chromosomes and other complex rearrangements

In addition, more complex rearrangements were also observed. Parent-of-origin typing revealed chromosomes with multiple de novo DNA breaks during the first cleavages (n = 17; in 6 embryos; 25%; 6/24) (Fig. 2C,2D). These rearrangements could involve both parental alleles or a single allele of a particular chromosome. No significant difference between the amount of rearranged paternal or maternal alleles was noticed (Fig. 2C,2D).

Chromosome 1 in blastomere "c" of embryo 20 had lost both ends of the maternal allele and thus may have formed a ring chromosome. Chromosome 10 in blastomere "d" of embryo 10 lacked both parental alleles of a p-terminal segment and the q-arm, while the intervening amplified DNA segment was paternal in origin. Hence, this blastomere lacked the maternal chromatid, while the paternal allele was broken at a p-and pericentric q-locus. The amplification amplification by an odd number of sister chromatid exchanges. Chromosome 6 in blastomere "c" of embryo 14 carried an intact paternal allele as well as a paternal duplicated segment lacking large chunks of both the p-and q-arm, suggesting ring chromosome formation by breakage of one paternal allele of a uniparental isodisomy. Other complex rearrangements involving multiple de novo DNA breaks per chromosome, including frequent (peri-)alphoid DNA-breaks, were detected on both parental alleles and are shown in Fig. 2C-E.

Microarray-guided FISH further corroborated such complex rearrangements in human cleavage stage embryos. SNP-array analysis revealed a p-and q-terminal deletion as well as an amplification of the intervening piece for chromosomes 6 and 7 in one blastomere of an embryo (Fig. 4A,B). In the nucleus of sister blastomere "e" only one signal for the p-and q-terminal FISH probes and two signals for the centromere for both chromosomes 6 and 7 were detected. This, as well as 7q-breakpoint flanking FISH, confirmed both rearrangements. In the remaining FISHed nuclei, a normal copy number for chromosome 6 was detected, while chromosome 7 was normal in two blastomeres and monosomic in two other blastomeres. Furthermore, in the arrayed blastomere a homozygous 18pter-deletion was also detected, while the remainder of this chromosome was hemizygous. A 4 th and 5 th FISH cycle confirmed the rearrangement (Fig. 4C).

The probes for 18pter as well as the centromere of chromosome 18 produced aneuploid FISH signals consistent with a homozygous 18pter-deletion in blastomere "a", an 18pter-truncated chromatid in blastomere "e" and an 18pter-amplification in blastomere "d". Based on the location of the FISH signals in the nuclei a break on chromosome 18 was also apparent in blastomere "b" (Fig. 4C). The data for additional chromosomal rearrangements are presented in Supp. Figure S1.

Discussion

Breakage-fusion-bridge (BFB) cycles are a hallmark of tumors and drive amplifications of oncogenes [Bignell et al., 2007]. However, these rearrangements also underlie the origin of inverted duplications associated with a distal deletion, which is a frequent type of chromosomal rearrangement in patients with birth defects [Zuffardi et al., 2009]. With the advent of array-CGH technology for pre-and postnatal genetic diagnosis, this type of rearrangement has proven to be much more frequent than previously estimated [Ballif et al., 2003;Zuffardi et al., 2009;[START_REF] Vera-Carbonell | Molecular characterization of a new patient with a non-recurrent inv dup del 2q and review of the mechanisms for this rearrangement[END_REF]. Also, ring and translocation chromosomes that contain inverted duplications flanking the breakpoint have been identified [START_REF] Rossi | Duplications in addition to terminal deletions are present in a proportion of ring chromosomes. Clues to the mechanisms of formation[END_REF]Zuffardi et al., 2009]. The latter most likely result from inv dup del chromosomes that circularize or translocate for stabilization of the uncapped chromosomal end. The generally accepted origin for inv dup del chromosome creation involves a breakage-fusion-bridge (BFB) cycle through either ( 1) meiotic dicentric chromosome formation between sister chromatids or homologous chromosomes because of chromosomal breakage and illegitimate repair or because of homologue misalignment during pairing at meiosis I and subsequent non-allelic homologous recombination (NAHR) between inverted repeated sequences, or (2) pre-meiotic iso-dicentric chromosome formation between sister chromatids because of chromosomal breakage and non-homologous end joining (NHEJ) with the replicated but broken sister chromatid, or because of chromosomal breakage and intrastrand annealing [Ballif et al., 2003;[START_REF] Vera-Carbonell | Molecular characterization of a new patient with a non-recurrent inv dup del 2q and review of the mechanisms for this rearrangement[END_REF]. Following the BFB-cycle, the rearranged chromosome is stabilized by telomere healing or capture, or by circularization, resulting in gametes with a chromosomal inv dup del scar. Previously we postulated the formation of isodicenctric chromosomes and suggested that breakage-fusion-bridges would occur in post-zygotic cells (Fig. 3A) which would be the origin of inv dup del chromosomes [Vanneste , 2009a]. In this study we prove those predictions (Fig. 3B-D) and show that the nonrecurrent inv dup del chromosomes are likely to result from postzygotic chromosomal breakage and isodicentric formation.

Parent-of-origin typing and microarray-guided FISH in blastomeres also revealed other complex chromosomal rearrangements. We show rearrangements involving multiple DNAbreaks on the same allele and provide ample evidence for (peri-)centric instability or centric fission resulting in i(p) or i(q) chromosome formation in human cleavage stage embryogenesis (n=16; 8 embryos; 33.3%). Centric fission has been characterized as an important driver of karyotype evolution [Perry et al., 2004] and i(p) and i(q) formation is an important mechanism in UPID etiology [Kotzot, 2001]. Hence, this study identifies chromosome instability in the cleavage stage human embryo as a major source of non-recurrent chromosomal disorders in addition to aberrant rearrangement and missegregation of chromosomes in pre-meiotic or meiotic germ cells. Furthermore, since this study includes cleavage stage embryos of good morphology, the data suggest that morphological scoring of a cleavage stage embryo following IVF is poorly suited to select cleavage stage embryos without genetic rearrangement. These data are also in line with the growing indirect evidence of CIN in the in vivo conceived early human embryo [Pflueger S., 1999;Macklon et al., 2002;Bruder et al., 2008;Conlin et al., 2010;Mkrtchyan et al., 2010;Rodriguez-Santiago et al., 2010]. For instance, common genetic mosaicisms for copy number as well as copy neutral anomalies that encompass large loci in the human genome are found in more than 1% of the individuals [Rodriguez-Santiago et al., 2010].

Yet, the high rates of de novo numerical and structural chromosomal aberrations that are formed during cleavage stage embryogenesis intuitively suggest that much more birth defects should occur. However, many of the cells with a rearrangement will be selected against because the altered gene content does not allow survival or efficient proliferation of the cell. In addition, the Deleted: [Vanneste et al., 2009] Deleted: i Deleted: is Deleted: [Bruder et al., 2008;Conlin et al., 2010;Macklon et al., 2002;Mkrtchyan et al., 2010;Pflueger S., 1999;Rodriguez-Santiago et al., 2010] F o r P e e r R e v i e w 18 majority of the cells with a numerical or structural anomaly contain not only additional chromosomal aberrations but also additional sister blastomeres with multiple chromosomal aberrations further decreasing the chance of cell or embryo survival. Only 3 out of 41 chromosomally abnormal blastomeres (derived from embryos for which at least 2/3 of the blastomeres were informative in this study; 62 blastomeres tested in total) contained a single chromosomal anomaly. Selection mechanisms against aneuploid cells have been uncovered. De novo aneuploidy in somatic cells can diminish their capacity to propagate in culture by a p53dependent cell cycle delay which converges cell populations toward an overall diploid karyotype [Thompson and Compton, 2010]. Furthermore, human embryonic stem cells have been found to initiate caspase-dependent apoptosis of chromosomally abnormal cells upon differentiation, therefore suggesting that differentiation is a mechanism that selects for diploidy [Mantel et al., 2007]. Fifty percent of the cleavage stage embryos in our studies do carry at least one blastomere which is normal diploid [Vanneste et al., 2009a]. Dependent on which blastomere(s) populate the embryo proper, postzygotic de novo chromosome aberrations are likely to cause constitutional chromosomal aberrations in a fraction (mosaic) or all the cells of the newborn.

Understanding the factors that cause the chromosomal breakages preceding the rearrangements seen in early embryogenesis would provide insight into the etiology of the low human fecundity, genetic disease development and genome evolution and would contribute to a better understanding of the low success rate to improve baby-take-home rates by FISH-based preimplantation genetic diagnosis for aneuploidy screening (PGS) when performed on the cleavage stage embryo [Vanneste et al., 2009b]. Many reports indicate that DNA-breaks are common in ejaculated human spermatozoa [Derijck et al., 2007;Aitken and De Iuliis, 2010] and it is hypothesized that human sperm DNA damage is a major contributor to poor rates of conception, impaired embryonic development, increased incidence of miscarriage and genetic [Aitken et al., 2009]. However, 35 paternal alleles as well as 23 maternal alleles were damaged by at least one chromosomal break during early embryogenesis in this study and the majority of these structural variants only appear in a subset of sister blastomeres suggesting that the DNA-breaks are instigated post-first cleavage division. Hence, sperm DNA-breaks or other forms of sperm chromatin damage are unlikely to explain the vast majority of de novo structural variants instigated during early embryogenesis.

Our data further question the clinical benefit of sperm DNA-testing which is performed in many fertility clinics to predict the success rate of the assisted reproductive technology that is offered to the couple [Sakkas et al., 2007;Aitken et al., 2009;Zini and Sigman, 2009]. Although a number of reports claim an association between sperm DNA damage and an increased risk of pregnancy loss in couples undergoing IVF or ICSI, there is no consensus and the impact of damaged sperm DNA on reproductive outcome and human development in general remains largely unknown [Sakkas et al., 2007;Aitken et al., 2009;Zini and Sigman, 2009]. Nevertheless, the number of embryos analyzed in this study is relatively small and it remains possible that sperm specific or male specific increases in paternal rearrangements would become apparent in embryos from couples with fertility problems. Application of the parent-of-origin typing developed in this study to each cell of cleavage stage embryos derived from fertile versus infertile men with a high sperm DNA fragmentation index might shed light on whether such paternal factors exist.

Many other mechanisms can underlie the chromosome breakages triggering the cleavage stage CIN. Upon fertilization it is generally accepted that the mature metaphase II (MII) oocyte delivers all the mRNA and proteins that are required for early cleavage cell divisions, as the human zygotic genome is only activated at the 4-to 8-cell stage [Braude et al., 1988] and sperm is largely void of transcripts [Ostermeier et al., 2004;Lalancette et al., 2008]. One possibility is Deleted: [Aitken et al., 2009;Sakkas et al., 2007;Zini and Sigman, 2009] Deleted: [Aitken et al., 2009;Sakkas et al., 2007;Zini and Sigman, 2009] Deleted: the Deleted: [Lalancette et al., 2008;Ostermeier et al., 2004] 1 for color interpretation. The copy number interpretation bars have been added to the parent-oforigin output (at y=0). ). Parent-of-origin indicates that the paternal allele is broken in blastomere "g". A paternal q-terminal fragment is lost and two identical alleles of the remaining broken paternal chromatid are retained in this blastomere. This suggests that an isodicentric chromosome accompanied by a terminal deletion is present in blastomere "g". Consistent with the breakage of the paternal allele, blastomere "d" contains an amplification of the reciprocal paternal q-terminal deletion in blastomere "g", while blastomere "c" carries a maternal monosomy which is consistent with a monopolar segregation of the isodicentric chromosome to blastomere "g".

These copy number and parent-of-typing profiles confirm the model proposed in (A). The slight difference between the position of the breakpoint of the 4q-terminal deletion in blastomere "g" and the position of the breakpoint of the 4q-terminal amplification in blastomere "d" is most likely due to the inability of spot-on breakpoint mapping in the applied single-cell copy number analysis. (E) Color legend to panels (B) and (D). The copy number bar interpreted from the microarray analysis of one biopsied blastomere, followed by a chromosome ideograph and the physical position as well as the color-label (SO: Spectrum Orange in red; SG: Spectrum Green in green; SA: Spectrum Aqua in light blue) of the FISH probes are shown on the left. Five FISH rounds were performed on this embryo which are depicted in consecutive order from top to bottom. The complex chromosomal rearrangement (B) SNP-array analysis of one blastomere of embryo 40 revealed a complex chromosomal rearrangement for chromosome 2 consisting of a duplication of the entire short arm, an additional q-terminal duplication and a normal copy number state for the intervening segment of this chromosome. Consistent with this rearrangement detected by SNP-array, we discovered two nuclei each revealing only one FISH signal for the p-and q-terminal probes, but two FISH signals for the centromere of chromosome 2. Hence these nuclei lack the reciprocal p-and q-terminal segments which are detected in excess in the arrayed blastomere, but retain the centric fragment. In contrast, another nucleus reveals no FISH signals for the centromere of chromosome 2, but two signals of the p-terminal segment and one signal of the q-terminal segment. One nucleus reveals 2 FISH signals for p-terminal and centric probes, but multiple signals of the q-terminal probe. Collectively, these FISH signals corroborate the complex chromosomal rearrangement detected by SNP-array analysis of the sister blastomere.

In addition, one nucleus produces two signals for all FISH probes indicating it contains the normal amount of chromosome 2, while in one nucleus no FISH signals for none of the probes could be detected suggesting it is nullisomic for chromosome 2. Human Mutation

  2005. Patients provided written informed consent for the collection of samples and subsequent analysis.

  for being transferred or frozen. Of these 13 embryos, 12

FISH

  Fluorescence in situ hybridization (FISH) BAC DNA was amplified by DOP-PCR and the DNA was directly labelled by the Random Prime Labeling System (Invitrogen, Carlsbad, CA).

  likely resulting from a meiotic non-disjunction further demonstrated that the algorithm pinpointed the affected allele. In embryo 19 all six blastomeres with monosomy 20

  simultaneously or after PGD. Subsequently, the breakpoint(s) of the structural chromosomal anomaly detected in the arrayed blastomere was investigated by breakpointflanking FISH on the remaining blastomeres. In one blastomere of embryo 33 a q-terminal duplication on chromosome 1 was detected by microarray. Using differentially labeled FISHprobes separated by approximately 100kb and flanking proximal to the 1q-terminal breakpoint, which was determined by SNP-array analysis, as well as a centric FISH probe for chromosome 1, scars of a BFB-cycle were detected in a sister cell. One nucleus had two centric FISH signals, but three signals of the breakpoint flanking probes, of which four signals (two of each probe) were in close proximity (Fig.

  paternal segment can be explained by ring formation and subsequent

Figure 1 .

 1 Figure legends

Figure 2 .Figure 3 .

 23 Figure 2. SNP-copy number and parent-of-origin profiles of complex rearrangements. Profiles characteristic of (A) isodicentric chromosomes, (B) inverse duplication-deletion chromosomes, (C) very complex rearrangements, (D) ring chromosomes, and (E) complex rearrangements encompassing chromosomal arms are shown. For each complex rearrangement, a chromosome ideograph, a bar representing the SNP-copy number interpretation (CN) and a bar representing the parent-of-origin interpretation (PO) is indicated from left to right. The color legend is depicted in (F). The embryo number (E), chromosome number (Chr) and affected blastomere are also indicated left of each chromosome ideograph. For each complex rearrangement, a table that describes the total number of detected copy number alterations and the parental origin of the affected allele is depicted. P: paternal; M: Maternal; N/D: Parent-of-origin not informative; S: Gain of both parental alleles; HoDel: Homozygous Deletion; UPID: Uniparental isodisomy.

Figure 4 .

 4 Figure 4. FISH profiles of complex genetic rearrangements in human cleavage stage embryos.

Formatted

  analysis of one blastomere is confirmed by FISH analysis of the remaining sister blastomeres. (A) pter and qter deletions on chromosome 7, (B) pter and qter deletions on chromosome 6, (C) pter homozygous deletions on chromosome 18 are confirmed by FISH. The amount of interpreted FISH signals is depicted below each nucleus as well as a chromosome ideograph according to the detected FISH signals. (*)(**)(***) For nucleus "e" in panel A (*), panel B (**) and panel C (***) the overlapping signals indicated by the arrow were interpreted as a FISH artefact. (****) One SA signal for nucleus "e" in panel C was considered unreliable (arrow) because of the artifact depicted in (*, ** and ***). ( + ) For nucleus "c" in panel B one additional SG-and SA-FISH signal is masked by the large stain (arrow). ( ++ ) FISHartefacts are indicated with an arrow. ( +++ ) For nucleus "c" in panel C one SA-signal (arrow) is considered unreliable because of the stain in the previous FISH cycles ( + and ++ ). (°) For nucleus "d" in panel C the SG-FISH signals were too weak to be visualized.

  **) For nucleus "e" in panel A and B the overlapping signals indicated by the arrow were interpreted as a FISH artefact. Deleted: (***) Deleted: (****) For nucleus "c" in panel C one SA-signal (arrow) is considered unreliable because of the stain in the previous FISH cycles.

F

  Figure 1 218x78mm (600 x 600 DPI)

Formatted:

  Font: Times New Deleted: The observation of 3 nuclei with four 5q and two 5p FISH-signals suggests that the two additional 5qfragments might have been covalently linked by NHEJ.

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Table 1 . Rules for parent-of-origin scoring

 1

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	o
	r
	P
	e e r
		R e v i e w

Table 2 . Affected alleles in whole chromosome imbalances uncovered by parent-of- origin analysis

 2

	Aberration	Total	Paternal allele	Maternal allele	No allele
			affected	affected	discrimination
					possible
	Nullisomy	9; 100%	9; 100%	9; 100%	-
	Monosomy	54; 100% 32; 59.3%	18; 33.3%	4; 7.4%
	Trisomy	90; 100% 18; 20%	22; 24.4%	50; 55.6%
	Tetrasomy or more 11; 100% 2; 18.2%	1; 9.1%	8; 72.3%*
	*Represent most likely amplifications of both maternal and paternal alleles

Table 2 : Semen characteristics

 2 +" indicates that at least one embryo related to the semen characteristics has a whole chromosome aberration b a "+" indicates that at least one embryo related to the semen characteristics has a segmental chromosome aberration c numbering of the embryos is described in Materials and Methods

		1	13 pericep-q Spectrum Orange	self labeled mix	RP11-139J24	RP11-304L17	RP11-535A03	RP11-429L04
	47	1	13q	Spectrum Green	self labeled mix	RP11-771H11 RP11-158K19	RP11-3P08
		1	8p	Spectrum Orange	self labeled mix	RP11-12F03	RP11-491C03	RP11-784G19
		1	8q	Spectrum Green	self labeled mix	RP13-733G14 CTD-2360M10	CTD-2192A17	CTD-639O03	RP13-655G18
	48	1	CEP 8	Deac	Vysis

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

We thank all families who participated in the study, the LUFC for the technical assistance, and R.

Thoelen for her help with the FISHs. This work has been made possible by a IWT (SBO 60848), FWO grant G.0320.07 and the SymBioSys Center of Excellence (Research Council, K.U.Leuven, EF/05/007) to J.R.V. E.V. was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Ethical Committee of the University Hospital Leuven

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Supp. Table S1: FISH probes that were used in this study.


1. Although the Chechen side understood the agreement as a recognition of its independence, the determination of Chechnya's status was postponed until 2001. 2. Brian Williams, "Caucasus Belli. The second Russo-Chechen war, 1999-2002. A struggle for freedom or sub-plot to the war on

Larzillière, Pénélope (2007) Chechnya: moving toward Islamic nationalism? in Blom, A., Bucaille, L. & Martinez, L. (ed.) The Enigma of Islamic Violence, London, Hurst, pp 89-104.

Chechnya : moving toward Islamic nationalism?

by Pénélope Larzillière Between the first Russo-Chechen war of independence and the resumption of the conflict in 1999, the range of actions employed by the Chechens evolved, as did the intensity of Russian army operations. While ambushes against the latter have remained the Chechen combatants' principal mode of action, recent developments in the conflict have been marked by the emergence of suicide attacks. The first war ended with the signing of the Khassaviurt accord in August 1996 1 , following the serious setbacks experienced by the Russian army. A treaty was signed in 1997 prohibiting the use of force in relations between the two countries. However, the situation in an independent Chechnya turned out to be difficult. The shattered economy left between 80 and 90 % of the working population jobless 2 . Russian promises of reconstruction in fact resulted in the economic isolation of Chechen territory through checkpoints set up all along the border. On the domestic scene, elections were organised in 1997. The candidates who were the most intransigent towards the Russians, and who had Islamist leanings, such as Basayev 3

On the Russian side, the second war was marked by far more extensive bombings, village "sweeps", the establishment of "filtration camps", ransom-and Udugov (Minister of Information under President Dudayev), were outnumbered by Maskhadov, the head of the Chechen army known for his willingness to compromise and negotiate with the Russians. Yet Maskhadov had great difficulty in establishing his authority over the whole of Chechnya, with certain warlords refusing to retreat. In October 1999, Dagestani and Chechen Islamist combatants, led by Basayev, made an incursion into Dagestan in order to provide assistance to Wahhabi villages. In doing so, they gave the Russians an excuse to invade Chechnya again. taking, etc. In addition to traditional guerrilla methods, in 2000 the Chechens began launching suicide attacks against the Russian army, as well as against the pro-Russian Chechen administration, for which Basayev generally claimed responsibility. The suicide attacks were strongly influenced by the idea of shahid, or Islamic ''martyr". Websites for many jihadist organizations readily portrayed Chechnya as one of the fronts where Muslims were being oppressed by kuffar, or "infidels", in this case Russians. The Russians immediately highlighted this state of affairs and made a direct link between such actions and Al Qaida-style international terrorism. The suicide attacks, rejected by the people, were then presented as the result of foreign influence, i.e. the "Wahhabis". Thus arose the issue of the ''Palestinization'' of the conflict, officially referred to for the first time in June 2003 by FSB (ex-KGB) director Nikolai Patrushev, as well as by certain journalists such as Anna Politovskaia 4

Islamism, an Islamic nationalist reinterpretation of the national struggle?

. But does the increase in suicide attacks in Chechnya mean the Chechen struggle has been co-opted by international jihadist movements? The theory we will develop here attempts to highlight the evolution of that struggle from nationalism to Islamic nationalism, as the discourse justifying the war is increasingly filled with religious references, while the issues at stake have remained nationalist. Such references are also historical, as they were already present during the anti-colonial struggles in the 19th century. Moreover, although the use of suicide attacks is part of the evolution towards Islamic nationalism, this increase in sacrificial violence cannot be attributed to the Islamists alone. Indeed, the latter were very present during the first Chechen war when such actions were not employed. Other aspects must therefore be included in order to understand this change. We must be careful in our analysis in any case, as the inaccessible terrain makes it difficult to evaluate the real impact of references to martyrdom on the population and groups of combatants.

Although Islam has been present in the region since the 8th century, it was not massively established in Chechnya until the 18th, and was implanted, in the 19th century during the Caucasus wars 5 4. Note that while Palestine also appears as a front for jihad on jihadist websites, the reference to martyrdom that one can observe in Palestine itself has little to do with the martyrdom of transnational jihad, as it exists within the context of Islamic nationalism: Pénélope Larzillière, ''Palestinian 'martyrdom', the new face of failed nationalism'', in Alain Dieckhoff and Rémy Leveau (dir.), Israéliens et Palestiniens, la guerre en partage, Paris, Balland, 2003, pp. 89-116. As shown by Khosrokhavar, although suicide bombings are within the range of actions taken by Islamic nationalists and international jihadists, their frames of reference for justifying such actions are different: nationalism regarded as sacred in one case, and the transnational umma (community) of believers in the other (Farhad Khosrokhavar, Les Nouveaux Martyrs d'Allah, Paris, Flammarion, 2002). 5. See Mikhaïl Roshin, ''Islam in Chechnya'', in Frédérique Longuet-Marx (dir.), Tchétchénie, la guerre jusqu 'au dernier, Paris, Mille et Une Nuits, 2003, p. 118. . The relationship between Chechen Islam and the anti-colonial wars constitutes an important point in understanding how the Chechen nationalist struggle was to be linked to Islam later on. That form of Islam was rooted in two Sufi brotherhoods: the Naqshbandiyya and the Qadiriyya. Every Chechen is associated with one of the branches of these brotherhoods, and members of the same clan may belong to different ones. These brotherhoods are also a form of social organization. Nevertheless, they lost a great deal of influence during the Soviet era, when Chechen society took part in the overall movement towards secularization. Chechen Islam, many of whose leaders disappeared, in particular during the deportation in 1944 6 , gave up many of its political aspects at that time and was transformed through an emphasis on Sufi rituals and traditions such as festivals, zikr 7

In addition to the emergence of jihadists , etc. Respecting those rituals during the deportation played a major role in maintaining Chechen identity.

8 in the Russo-Chechen conflict, which we will examine later on, one issue of great importance involves the response to Salafist religious and political ideas, in direct opposition to local Sufism. Two aspects played a crucial role: a generational conflict on the one hand, and a connection to the national struggle on the other 9

Claiming a connection to Wahhabism has enabled young people to challenge clan-based decision-making structures. Through such references young people have succeeded in legitimizing their decision-making capacity. Within the Palestinian context, Islamism has challenged patriarchal authority by referring to a higher source of legitimacy and by establishing a direct reference to the Koran (in essence, playing the Koran off their fathers). Similarly Wahhabism, by challenging Sufism and especially social structures connected to it, has provided an opportunity for young people .

The generational conflict 10 6. On 23 February 1944, Stalin deported the Chechens to Kazakhstan, accusing them of collaborating with the Nazis. The memory of that deportation has remained central to the Chechens. 7. A group dance accompanied by prayers and songs. 8. By the term ''jihadist'' we are referring to international Islamist groups supporting the Salafist doctrine and associating it with jihadism. By ''Wahhabis'' we mean local Islamist groups supporting Salafism. 9. In this regard it could prove interesting to compare it to the role played by political Palestinian Islam as seen in movements such as Hamas. Indeed, the identity issue serves the same function here of reinterpreting the national struggle and of challenging community decision-making structures. 10. Chechen Committee, Tchétchénie, dix clés pour comprendre, Paris, La Découverte, 2003, p. 37. . It has highlighted a direct connection to God, without any intermediary religious authorities, and built a religious relationship which can take on a more individualistic side in certain contexts, beyond the highly prescriptive rules of that movement and its organization in more or less closed groups. Thus the Wahhabis have often found themselves in direct opposition to village councils. Furthermore, this new religious reference has gone hand-in-hand with the creation of armed groups, which has enabled them to impose their decisions through the use of force. Lastly, in a devastated economy, these groups have provided a great deal of financial assistance. Social advancement has been possible within these movements through knowledge about Islam. Chechen Sufism has suffered from its failure to transmit such knowledge. Thus, in particular since the institution of Sharia, individuals with expertise on the subject of Islam -acquired over a few years through ad hoc training in Arab countries, including a certain familiarity with the language of the Koran -can attain positions upon their return that were previously unthinkable (president of a court, for instance). However, for the time being we lack sufficient information about the sociological profiles of those supporting Wahhabism. The challenge to the established order has therefore come about through the use of weapons, but also through this reference to knowledge, even if the latter is marked by intense ideology.

The relationship to the national struggle A new interpretation of the national struggle has also been facilitated by the reference to Islamism. Indeed, during the second Russo-Chechen War some of the Sufi authorities -in particular Kadyrov, a former mufti and combatantwere gradually discredited as the war progressed and repression by the Russians got worse, since they had collaborated with the latter. Although the administration has altered certain attitudes since then, it has continued to apply pro-Russian policies. On the opposite side, combatant groups such as those led by Basayev have linked Islamism to the national struggle and referred to the figure of the Imam Shamil. The latter, who belonged to the Naqshbandi brotherhood, founded an independent imamate in the 19th century uniting Chechnya and Dagestan where he established the Sharia. He was defeated and deposed by the Russians in 1859. The rooting of Islam within the national struggle has also been achieved through the notions of ghazavat and jihad. While jihad refers to ''the believer's efforts to improve himself'' (major or inner jihad), or to the ''holy war carried out to defend Muslims and their lands'' (minor or outer jihad), ghazavat specifically designates combat between armies 11

The notion of jihad developed here is thus closely tied to references to independence and has only acquired real meaning for certain Chechen combatants within the context of the national struggle. In that sense one may speak of Chechen Islamic nationalism. By referring back to a past involving Islamism and the independence movement, Chechen Islamists are adapting jihadist Islamism to new territory. The creation of an Islamist state could then . It was first used in reference to the 19th century Caucasus Wars, and later during the first Russo-Chechen war.

11. Alfred Morabia, Le Gihad dans l'islam médiéval, Paris, Albin Michel, 1993, pp. 96 and 110. appear to be an objective which, by reinforcing Chechen society, will also promote its autonomy. In the same way that the idea of jihad developed by Palestinian Islamists suggests not so much a religious agenda as a way of incorporating the national struggle into a religious eschatology -the main objective remaining the territory's independence -, it seems that for the Chechens this notion should be interpreted more in the context of the past and its glorification. Furthermore, the inclusion of the national struggle within this set of references links this specific form of resistance to other examples of resistance through a crisscrossing chain of identification which has helped Chechnya break out of its isolation, both symbolically and in its attempts to raise funds.

The limited impact of internationalist jihadist groups

For certain -mainly Arab -international jihadist groups, Chechnya is a substitute for Afghanistan. The ideology developed by such groups has turned the Russo-Chechen conflict, like Palestine, into a zone of opposition between oppressed Muslims and infidels. However, they remain very limited in number in relation to the entire Chechen resistance movement; and while their propaganda routinely refers to Chechnya, it is mainly aimed at an Arab Muslim public. The extent of their impact has depended on links to local combatant groups and on territorializing their references, in other words on how well they adapt to what is at stake locally.

The impact and tactics of armed Wahhabi groups in Chechnya

The first jihadist groups arrived in Chechnya during the first war between 1994 and 1996. Before their arrival, the neo-fundamentalist Wahhabi doctrine had spread in Dagestan under the influence of a Dagestani preacher named Kebedov 12 . Dagestani villages had turned Wahhabi and adopted the Sharia. The term Wahhabi, referring to Wahhabism 13 12. Mikhaïl Roshin, chapter cited, p. 138. 13. Wahhabism grew out of an alliance between a tribal clan, the Sauds, and a religious reformer, Abdul Wahhab (1703-1792). See Olivier Roy, L'Islam mondialisé, Paris, Le Seuil, 2002, p. 136. , a highly conservative form of Sunnism and the official religious doctrine of Saudi Arabia, is often rejected by such individuals themselves, who see it as an oversimplification of their doctrine and too specific a link with Saudi Arabia. The term Salafist is generally used by jihadist groups, although it is not certain that this is widespread in Chechnya. Salafism is an idea that was created in the late 19 th century. The term itself comes from salaf (''pious ancestor'') andthe trend grew out of a desire to return to a purer form of Islam by referring directly to the original texts and by adopting the same lifestyle as the prophet. This ''return'' had a political dimension through its resolve to challenge colonialism 14 . Political positions within the Salafist movement are extremely varied, with the jihadist faction merely one of its more extreme forms 15 Thus the Wahhabis firmly established themselves through their military successes, and their Islamist discourse was only meaningful in reference to the struggle. For a Chechen combatant, choosing an armed group was more a function of proximity and renown than of ideological orientation. Their relationship to religious practices fluctuated . Theories attempting to explain how such doctrines took root usually highlight the usefulness of a form of identification allowing for differentiation from and opposition to the Russians in the post-Soviet context. With respect to Chechnya, there are two different dimensions through which these groups have taken a position in local conflicts and thereby gained an impact -through their contribution to the national struggle and their challenging of traditional structures in organizations they were representing.

Integration in the struggle against the Russians Indeed, these combatants perceived above all as foreign extremists by the Chechens, were not at all well liked at first. Nevertheless, their alliances with Chechen warlords, the most famous being the one sealed between Basayev and Khattab, a Saudi Arabian Wahabbi and former combatant in Afghanistan, have afforded them a certain measure of integration. This alliance clearly shows their determination to join together with effective groups and to obtain outside financing. As for Khattab, this local ''sponsorship'' was essential for him because the involvement of his combatants was not well accepted by the population. However, his success against the Russians in the field strengthened the position of his jihadists, and from that time on certain Chechen combatants began making Islamist references. Subsequently, their successes were widely broadcast through videotapes. During the first war, Basayev's most famous military exploit was the February 1995 hostage-taking following a sortie which aimed to break through the Russian army encircling the Chechens and advance two hundred kilometres to the north of Chechnya. During negotiations the Russian Prime Minister Chernomyrdin announced the end of Russian operations. Although the ceasefire did not hold, it gave the Chechen resistance time to take in the new combatants from the villages destroyed by the Russian army. That type of operation gave particular visibility to this group of combatants, who nevertheless constituted a minority among the Chechen troops led by Maskhadov. They are estimated to have been three or four thousand in number during this period. 16 14. Olivier Roy,op. cit.,p. 133. 15. On this point,Olivier Roy,op. cit.,p. 141. 16. Chechnya Committee,op. cit.,p. 36 . And the opposition between Sufism and Salafism, which was nevertheless one of the things at stake for foreign combatant groups, held little meaning for the young people who joined such groups and found themselves surrounded by Islamist references linked to the national struggle.

Challenging local authorities?

More or less united with the rest of the Chechen resistance during the first war, these Islamist combatant groups were involved in the extreme division of power in post-war Chechnya. Indeed, their position was threatened within Chechen society. The population favoured negotiating with the Russians and was eager to believe in the accords signed. As the Islamist's presence in the Chechen resistance was disproportionate to their real impact on the population, the end of the war signalled a loss of power for them. The desire to maintain their position manifested itself through two forms of opposition, on the one hand within the independence movement against Maskhadov, who vacillated between compromise and rejection, and on the other hand against the traditional authorities and the Sufi hierarchy. Their position here was not so much a specificity of the Chechen Islamist movements as a standard attitude of warlords eager to maintain their power by carving out fiefdoms -sometimes linked to the criminal world -within which they refused to obey the main authority, in this case Maskhadov. In parallel to that refusal, the Islamists also opposed the local Sufi authorities. As for the authorities themselves, they struggled against the combatants, challenging the strength they derived from taking part in the war. They sought to keep them out of the villages to avoid Russian reprisals against civilians. In general they developed an attitude of compromise with the Russians rather than armed struggle, illustrated particularly well by the fate of Kadyrov, the former mufti who ran the pro-Russian Chechen administration from June 2000. In that sense, the authorities constitute a form of opposition to the Islamic nationalists. For the jihadists, there is the added desire to reform Sufi Islam, considered degenerate by comparison with a pure, original Islam. This opposition led to violent internal clashes in July 1998. Within this context, Maskhadov tried in vain to expel the Wahhabis from Chechnya. He obtained no assistance from the Russians. The Chechen Islamists then proceeded to carry out what could be called both an ideological and a strategic U-turn by strengthening their alliance with the Dagestani Wahhabis. An Islamist congress of Dagestani and Chechen Islamists had been formed in 1997. In 1998 it created an armed division to be led by Basayev. Ideologically speaking, it manifested far greater determination to establish an imamate connected to Shamil. In strategic terms this alliance highlighted a desire for opposition, and clearly led the Islamists to overestimate their influence on the people. Furthermore, the ties between Dagestan and Chechnya prompted them to take into account other aspects at stake such as the conflict between the Wahhabis and the Dagestani government. In August 1999, several hundred combatants commanded by Basayev and Khattab entered Dagestan to ''liberate'' it and help form an Islamic state united with Chechnya. But the Islamists greatly underestimated the people's opposition to their movement and were faced with local resistance in addition to attacks by the Russian army. That incident marked the most extreme point in the gap between the Islamists' ideas and the Chechen population. While striving to maintain their position, the Islamists were forced to take things even further. They had a double interest in the situation in Chechnya remaining unstable. First, their influence in the resistance movement -due to their considerable resources -gave them a more important role to play. Secondly, their ideology was only meaningful to people during times of conflict, particularly when conflict intensified and there appeared to be no other way of settling the situation. Beyond their effective participation in armed groups, what was the Islamists' exact ideological posture? Which ideological discourse?

One cannot talk about one Islamist discourse in Chechnya, since there are several of them dealing with different issues. Roughly, one could say that references to Islam in Chechnya are threefold: a traditional Sufi Islam which is also involved in the organization of society, an Islamic nationalism, and a jihadist Islamism backed by foreigners. The three discourses are different, as are the leaders of these three trends and their positions on the national struggle. However, these distinctions are not necessarily meaningful to their rank-andfile militants who shift from one to the other. Despite the use of the same vocabulary, a distinct gap is apparent between the discourse developed around Chechnya by the international jihadist movement and that of local Chechen Islamists found on the internet, on videotapes, in declarations made by their leaders, etc. Nonetheless, while one may jump from a territorialized discourse on independence to a de-territorialized discourse with jihadists, the one constant always advanced is the notion of ''martyrdom'' and the capacity for sacrifice. For the jihadists, Chechnya has no regional or historical specificity and is merely a place illustrating the oppression of Muslims by kuffar. As in Palestine, it has been used a-posteriori to develop that theory and to legitimize their position by showing the struggle against the abuses of the Russian army in Chechnya. One can distinguish the ''non-specialized'' jihadist websites, in which Chechnya and Palestine constitute the central -but not the onlythemes, from those specifically concerned with Chechnya. In addition to the websites, Azzam, generally thought to be Al Qaida's publishing house, has brought out a CD-Rom on Chechnya in Arabic showing the wartime atrocities and Chechen ambushes against the Russians with a number of personal accounts and wills from mujahidin. Videotapes of ambushes led by Basayev and Khattab in Chechnya are also being circulated. This type of media is important because websites are often inaccessible.

In these documents, particularly in the wills of kamikazes and bequeathed videotapes 17 Female martyrdom is featured on the website www.qoqaz.com. Piety and the shahidats' capacity for sacrifice are highlighted: ''I know what I'm doing, paradise has its price and I hope it will be the price of paradise. Hawaa Barayev, the sword of jihad.'' A ten-page document explains the specific role women can play within the framework of jihad , the Russians are presented as kuffar. But the issue of Chechen independence is not the main theme. The videos, photographs and accounts stressing the horror of war, lingering over images of the dead and wounded, and showing the combatants in action are juxtaposed with verses from the Koran. Next to photographs of dead "martyrs" is the text: ''Do not think that those killed on the path of Allah are dead. On the contrary, they are living with their God, very well provided for.'' (Koran III,169). Similarly, the wills stress the relationship to Allah and jihad, with no mention of the Chechen question. 18 . This glorification of women is always to the detriment of men who have not joined the fight: ''A great many women are now taking part in jihad and I hope that the men will also participate in jihad and not take on the women's role by staying at home.[...] Our ancestors would have killed anyone who tried to touch their wives, but Muslim women are now being attacked and raped in front of those who call themselves men, and they care so little about the honour of their Muslim sisters that they sit there drinking tea while listening to such appalling news'' 19 In contrast to these general invocations, the documents shown on the kavkazcenter website run by the Chechen Udugov -who also has a television channel which he now runs from Qatar -, as well as those on ''martyrdom operations'', are in Russian rather than Arabic; and their discourse is centred around the need to liberate Chechnya. This is the specific framework within which jihad is referred to. One example is this declaration made by the hostage-takers at a Moscow theatre . 20 17. To quote only a descriptive summary of certain videos in Arabic: Chechnya, the destruction of a nation (December 1999) shows attacks against civilians, the systematic destruction of non-military targets by Russian forces, and amputated young children and babies; Massacre in Chechnya (October 1999) shows massacres perpetrated against women and children by Russian forces; Jihad in Dagestan shows attacks by the jihad in August and September 1999, when Chechen mujahidin went to help their Muslim comrades in Dagestan. 18. To take part in combat if the local commander decides the struggle is such that women must participate, to support the combatants in the battlefield, to raise mujahidin children, to engage in physical training, to urge loved ones to take part in jihad, and to study military medical practices. 19. Excerpt from a text presenting itself as an English transcription of a video entitled No Surrender ! It is a dialogue between two women, Hawwaa and Luiza, preparing to commit a suicide attack, and their instructor. The document, initially featured on the website www.qoqaz.com, can be found on www.terrorisme.net. This website, as well as www.religioscope.com, is run by Jean-François Mayer, a professor at the University of Fribourg. 20. On 23 October 2002 a Chechen commando took several hundred spectators hostage. Russian special forces stormed the building using paralysing gas. All members of the commando were killed, as were numerous hostages.

, broadcast on Al-Jazeera: ''We have come to the capital to end the war and attain martyrdom here through the path of Allah. We demand an end to the war and the withdrawal of Russian forces.[...] Every nation has a right to self-determination, but Russia has taken that right away from the Chechen people and today we want to take back that right granted us -and to all other nations -by Allah the Most Gracious, the right to freedom and self-determination. The Russian occupants have flooded our country with our children's blood and we have waited a long time for a fair solution [by appealing to] human consciences, but the world is living in a coma and doesn't care about the assassination of innocents.[...] That is why we have chosen this path -the path of jihad for the liberty of the Chechen people.'' 21 There are two opposing discourses. On the one hand, de-territorialized jihadism, and on the other hand, ethnic nationalism evolving towards Islamic nationalism. The two kinds of discourse are not intended for the same audience. Jihadist sites broadcasting mainly in Arabic and English are mainly targeting an Arab-Muslim audience . 22 . The same type of competition was found in Palestinian camps in Lebanon between Islamic nationalism -in movements such as Hamas or Islamic Jihad in this case -and the Salafists' jihadist Islamism. When the mosques were taken over by Islamic nationalist movements such as Hamas, it was perceived as a way of preventing jihadist movements from taking root23 An evolving range of actions and the increase in suicide bombings . Although somewhat modified by Islamic nationalism, Chechen nationalism's significant influence limits the extent to which jihadist ideology can take root in the country, even if some figures from this movement are present. Islamist ideas are only meaningful for them through an association with the national struggle and its reinterpretation.

Is the evolution towards Islamic nationalism a key element in understanding the adoption of a range of actions including suicide bombings? The latter use 21. A French translation of these declarations can be seen on www.terrorisme.net: Prise d'otages à Moscou (26 octobre 2002).

22. An example of the impact of this kind of discourse on Palestinian refugees in Lebanon in 1999, in the context of clashes with the Lebanese army, has been analysed by Bernard Rougier: ''A sizeable part of this group became literally immersed in jihadist Islamism, identifying itself with the population of Grozny suffering daily bombings by Russian troops. The timing and the place for the fighting were synchronised with the destruction of Grozny by the Russian troops, and in the feverish minds of those following the fighting in Aïn el-Héloué, the struggle against the Lebanese army was indeed an extension of the Checehn jihad against the Russian army.'' Bernard Rougier, Reconstructions identitaires et mobilisations religieuses dans les camps de réfugiés palestiniens au Liban depuis la fin de la guerre (1990-2001), thesis, Paris, Institut d'études politiques, 2002, pp. 354-355. A young Palestinian from the camp even committed a suicide attack against the Russian Embassy in Beirut (Rougier,ibid.,.

the shahid, or Muslim martyr, as a reference, and in that respect the link might appear quite clear. Suicide bombings are more characteristic of the second war, particularly in 2003. From 2000 to 2003, the Russo-Chechen war was defined on the Russian side by an intensification of methods of massive repression 24 as well as widespread corruption. On the Chechen side, the resistance movement was divided. The people were at the end of their tether. The legitimacy of the pro-Russian administration, already very weak, was not strengthened by the March 2003 referendum, which was widely contested. Furthermore, the combatants clashed over which methods to employ. While ambushes and guerrilla warfare remained the main mode of action, bombings and suicide attacks increased despite Maskhadov's disavowal. Other kinds of operations associated with the idea of martyrdom had been carried out by the Chechens in the past. The main mode of action during the first war therefore consisted of ambushes against the Russian army. However, certain very highrisk attacks designed to help fighters break out of encirclement were presented as suicide attacks. Moreover, some combatants in Islamist battalions celebrated their funerals before going into combat 25 . Nevertheless, it cannot be called suicide in a literal sense except when volunteers were sent over minefields, as in Iran 26 , to allow the main combatants to get out of Grozny under siege. There, they were sure to die. In the two other cases, they used tactics aimed at turning apparently desperate situations around where there was still a chance for survival 27 It seems impossible to establish a direct causal link between the increase in suicide bombings and the evolution towards Islamic nationalism, or at least to attribute full responsibility to Islamist organizations in Chechnya. Since the resumption of the conflict, responsibility for the bombings generally was claimed by Basayev until his death in July 2006.. But two types of operations must be distinguished. Some, like the taking of hostages at the Moscow theatre, require a great deal of organization, while others -isolated acts with simple targets -seem to be carried out by relatively unorganized individuals . 28 24. On 11 April 2003, Le Monde published an article concerning a report from pro-Russian Chechen authorities sent to ''the highest level of State'' (i.e. Vladimir Putin). This unpublished document mentioned among other things the execution of one hundred civilians per month throughout 2002, nearly three thousand corpses found in mass graves, kidnappings and torture. On 17 April, the Chechen Prime Minister, Anatoli Popov, declared that it was not a ''secret report'' but rather the results of an inquiry led by the Chechen state prosecutor Viktor Kravtchenko. According to him, the text actually mentions three hundred civilian kidnappings involving Russian soldiers, some of whom are already serving time. 25. Brian Williams, ''Unraveling the links between the Middle East and Islamic militants in Chechnya '', Central Asia-Caucasus Analyst, 12 February 2003, p. 2. 26. Farhad Khosrokhavar, L'Islamisme et la Mort, Paris, L'Harmattan, 1995. 27. For Hamit Bozarslan, eliminating . It is unlikely, for example, that Basayev had any real hold over the latter. These acts are carried out by Chechens, and surely not by international jihadist militants. The first suicide attacks using truck bombs were perpetrated in July 2000; but the true intensification and exportation of this mode of action on Russian territory began in December 2002. Nonetheless, the Russian army in Chechnya and the pro-Russian Chechen administration were the targets of the majority of bomb attacks. This specificity highlights the extent to which Chechen society was divided. One of the bombings targeted Kadyrov, the head of the administration 29 These diverse elements tend to reinforce the theory of local appropriation of acts such as suicide bombings, which indeed developed through the notion of sacrifice for the cause, and the shahid, but they cannot be considered as the result of indoctrination based on the person's isolation. Moreover, Chechens tend to distance themselves from the jihadist discourse and consider the Islamic nationalists' violence as a form of revenge, the weak man's weapon. The suicide attacks committed by women are associated with the idea of ''sacrifice, not in the name of any doctrine, but as an act of revenge or a way of taking justice into one's own hands since one cannot count on the judicial system, nor on help from anyone. Isolation and abandonment by the international community are mentioned constantly.'' . 30 There are some striking analogies here to the case of Palestine, where prospective suicide bombers for the second Intifada (contrary to those in [1994][1995][1996], far from being seasoned militants from organizations, were recruited in a much more widespread fashion and often went to the organizations themselves when ready to take action. The same intensification of religious . The history of these women confirms this. People talk of ''war widows'', and there is always a deceased husband, brother or other relative to justify their desire for revenge. Although these women are increasingly religious, according to Bleuenn Isambard, ''the young women who died at the Nord-Ost Theatre in Moscow in October 2002, and others who blew themselves up against Russian targets -or tried to, more or less successfully -were for the most part ''modern'', educated young women who did not belong to extremist movements. Some were clearly ''manipulated'' or used as tools, while others decided on their own and made the move alone, or nearly so.[...] Those who were willing to commit such deeds -and even the acts themselves -were appropriated after the fact. The willingness to commit such acts is therefore not the result of indoctrination, but clearly the person's own decision, which is subsequently reinforced and supported, at times by those around him/her, and through religious interpretations stressing the value of sacrifice. The main motivation seems linked on the one hand to a desire to avenge a close relative, and on the other hand to the general context of the situation in Chechnya which is perceived as devoid of hope.

29. The bomb attack on 14 May 2003 carried out during a religious celebration seemed to be targeting Ahmed Kadyrov. There were fifteen deaths and one hundred and forty wounded. Sources disagree about whether it was a suicide attack by one or two women, or a bomb which had been placed inside a loudspeaker. 30. Interview with Bleuenn Isambard upon his return from Chechnya in the summer of 2003.

practices took place a few weeks prior to the event. But, contrary to what has occurred in other contexts, the organization did not have to isolate the individuals, who continued to live in their everyday environment up to the last day. As in the case of Chechnya, the suicide bombers in Israel are not recruited from a specific environment, and definitely not from a particularly poor or underprivileged one by the standards of the society as a whole. The Chechen and Palestinian cases are indeed similar, as they both contain broad social breeding-grounds which have been more or less exploited by these organizations, although not created by them. Despair resulting from international indifference must be taken into account as one source for such breeding-grounds.

In parallel with the increase in suicide bombings in Chechnya, the use of religious vocabulary has become more pronounced in talking about the conflict, along with the highlighting of combatants' religious practicesbeginning with the ''martyrdom'' of the suicide bombings. But this does not necessarily reflect any influence from international jihadism. First, one must underline the heritage of past struggles dating back to the 19th century, when Islam was closely tied to anti-colonialism. The use of references from international jihadism indicating Chechnya to be a hotbed ofkuffar oppression of Muslims is only meaningful if associated with the plan for national liberation in Chechnya. The Islamists' main impact has been through their battalions' exploits, and through a subsequent association -for the time beingwith a heroic (but also extremist) image. Conversely, their attempts to change values within Chechen society and their opposition to traditional structures have alienated a segment of the population. However, this may also afford them some credibility with the younger generation, to whom they represent a chance for social advancement and greater power. Although the reference to martyrdom provides a framework for legitimizing suicide bombings, these mainly grow out of a desire for revenge, of a personal nature in most cases -suicide attacks against the general who killed one's brother, for example -, and as such could potentially find wider support among the Chechen population. That same aspect of revenge -a list of names could be mentioned in a will -is present in Palestinian kamikazes; but injecting the notion of an eschatological jihad adds a temporal dimension which allows the lost national struggle to be recast with very long-term prospects for victory. That aspect seems to be missing from the Chechen equation, perhaps which perhaps confirms a weaker ideological link between suicide attacks and the Islamic nationalist reinterpretation of the struggle 31 31. It would have been impossible to write this chapter without the conversations I had with Bleuenn Isambard, Uwe Halbach, Anne Le Huérou, Aude Merlin, Frédérique Longuet-Marx, Silvia Serrano, Mairbek Vatchagaev and Laurent Vinatier, to whom I would like to extend my thanks.

.

  and Christoph Zuercher, Institutionen und organisierte Gewalt : Konflikt und Stabilitätsdynamiken im (post-)sowjetischen Kaukasus, p. 36.

  the idea of risk in suicide attacks, in which death is certain, is a crucial aspect in the development of sacrificial violence. Hamit Bozarslan, From Political Contest to Self-Sacrifice: Violence in the Middle East, Princeton, Markus Wiener Publishers, 2004. 28. Laurent Vinatier, ''Prospective reflections on the political evolution of Chechnya'', Round table discussion on Chechnya, EHESS-IISMM, 5 May 2003.

. Bernard Rougier, ''Religious dynamics and national identity in Palestinian refugee camps in Lebanon'', in Jean-Francois Legrain, (dir.), ''Waiting for Palestine'', Maghreb-Machreq, n 176, September 2003.

Pénélope Larzillière

Pénélope Larzillière is a researcher at the Institut de Recherche pour le Développement (IRD) and the author of Etre jeune en Palestine, Paris, Balland, 2004.