Hum. Reprod. Advance Access published online on October 21, 2008
Human Reproduction, doi:10.1093/humrep/den323
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Constitutional DNA copy number changes in ICSI children
1 Department of Obstetrics and Gynecology (internal postal code 791), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands 2 Department of Human Genetics, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
3 Correspondence address. E-mail: j.kremer{at}obgyn.umcn.nl
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Abstract |
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BACKGROUND: Over the last three decades, technological developments facilitating assisted reproductive techniques (ART) have revolutionized the treatment of subfertile couples, including men suffering from severe oligospermia or azoospermia. In parallel with the advent of these technologies, there is a great concern about the biological safety of ART. This concern is supported by the clinical observation that the frequency of congenital malformations is slightly elevated among ART-conceived children.
METHODS: In this explorative study, we have used tiling-resolution BAC array-mediated comparative genomic hybridization to investigate the incidence of de novo genomic copy number changes in a group of 12 ICSI children, compared with a control group of 30 naturally conceived children.
RESULTS: In 6 of the 12 ICSI children, we found 10 apparently de novo ‘same direction genomic copy number changes’ [i.e. simultaneous copy number gain (or loss) with respect to both biological parents], notably losses. In statistically significant contrast, similar observations were encountered only six times in the control group in 5 of the 30 children. However, our study group was small, so a larger group is needed to confirm these findings.
CONCLUSIONS: Loci at which we found de novo alterations are known from the human genome database to be prone to large DNA segment copy number changes. As discussed, various molecular mechanisms, including the consequences of delayed male meiotic synapsis and replication fork stalling at early embryonic cell cycles, might trigger these copy number changes.
Key words: ICSI/array-CGH/genomic polymorphisms/pyrosequencing/CNV-instability
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Introduction |
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In Europe, in 2002, the clinical pregnancy rate for ICSI was 29.4% per transfer, and it has been estimated that 1–3% of the overall number of live births are the result of ICSI (Andersen et al., 2006
). These figures clearly demonstrate that ICSI is currently being successfully applied on a large scale. Even men with azoospermia often have the possibility to father their own genetic progeny, through ICSI with surgically retrieved sperm at the level of the epididymis or testis.
In parallel with the advantage of these technological developments, there is an observation that ICSI seems to be associated with slightly elevated incidences of certain birth defects. Large follow-up studies by several independent research groups have revealed a small though statistically significant increase in the incidence of certain birth defects in IVF and ICSI children compared with naturally conceived infants (Hansen et al., 2002, 2005; Zhu et al., 2006). In theory, this increased risk may be caused by several factors which may either act alone or in conjunction (Patrizio, 1995; Kurinczuk, 2003). In the first place, male gamete-associated risks: sperm carrying DNA anomalies, i.e. breaks, aneuploidy, Y chromosome deletions or structural changes, may be transferred (Kremer et al., 1997; Bernardini et al., 2000; Morris et al., 2002). Second, there are risks contributed by the female gamete: the injection of the oocyte itself may cause damage to the ooplasm or meiotic spindle apparatus, and its DNA repair status may determine the mutagenic outcome of sperm DNA lesions (Bernardini et al., 2000; Kato et al., 2007; Marchetti et al., 2007; Derijck et al., 2008). Suboptimal female gametes, which otherwise would have been bypassed by natural selection, may be fertilized due to ovulation induction (Shi and Haaf, 2002). Indeed, it is beyond any doubt that a significantly higher rate of de novo chromosomal anomalies such as sex chromosome aneuploidies and structural chromosome anomalies, notably reciprocal translocations, has been observed in ICSI-mediated offspring (Bonduelle et al., 2002).
This has led to a situation in which we do not know to what extent assisted reproductive techniques (ART) increase the genetic load, how epigenetic aspects my be involved as well and how this could be reflected in the expressed congenital malformations. Lack of knowledge regarding the primary molecular mechanism(s) underlying the observed ICSI-associated increased rate of birth defects has thus far prevented further technical innovation for optimalization of the procedure.
Earlier results from array-based whole genome profiling in the fertile population have already firmly established that genomic duplications and deletions in the size range of a few kilobases to several megabases are relatively common (Iafrate et al., 2004; Sebat et al., 2004; Redon et al., 2006). These duplications and deletions are not necessarily translated into readily recognizable phenotypic changes, although a role in human evolution has repeatedly been suggested (Cooper et al., 2007; Jiang et al., 2007). At the same time, de novo regional duplications, deletions and inversions, triggered by genomically unstable sequences, involving ectopic homologous recombination are well-established causes for a still growing number of genetic diseases (Stankiewicz and Lupski, 2002).
In the present explorative study, we used bacterial artificial chromosome (BAC)-array mediated comparative genomic hybridization (array-CGH) analysis in order to identify constitutional de novo DNA copy number changes, including those that are phenotypically silent. With DNA copy number variation (CNV), two or multiple copies are arranged in tandem and/or there may be more elaborate patterns such as those caused by segmental duplications, where the numbers of copies are highly variable (Redon et al., 2006). In contrast to most of the other whole genome profiling platforms that were available at the time the study was performed, the tiling-resolution BAC arrays used provide unbiased genomic coverage, including polymorphic genomic intervals (comprehensively listed at the ‘Database of Genomic Variants’, http://projects.tcag.ca/variation/), at a practical resolution of around 100 kb (Krzywinski et al., 2004).
Through the use of BAC arrays, it is possible to detect DNA changes (i.e. large-scale copy number changes) more efficiently than by using the single nucleotide polymorphism (SNP)-arrays which were also available at the time this study was performed, because with the latter the CNV intervals were systematically underrepresented (Coe et al., 2007; Hehir-Kwa et al., 2007). This is of considerable importance since our data strongly suggest that copy number changes of large polymorphic intervals are frequently targeted in ICSI-children.
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Materials and Methods |
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Study design
This explorative study was performed with prior written informed consent of both parents and with approval of the Institutional Review Board of the Radboud University Nijmegen Medical Centre. Blood samples were obtained from both parents and from the umbilical cord of the child immediately after delivery. Six children (four boys and two girls) were born after ICSI with ejaculated sperm from fathers suffering extreme oligoasthenoteratozoospermia (OAT) in which no Y chromosome microdeletions or chromosomal abnormalities were found. Six additional children (two boys and four girls) born after ICSI with sperm from a percutaneous epididymal sperm aspiration (PESA) procedure had fathers with obstructive azoospermia [two times congenital bilateral absence of vas deferens (CBAVD) and four times failed vasovasostomy].
Relative genomic copy number profiles of all children were determined by separate comparison with both parents.
As a reference, we used genomic copy number profiles from 30 trios (child and biological parents), which were produced in the context of a previous departmental research program (de Vries et al., 2005), all without any apparent link to reduced fertility. In this study, the proband and both parents were individually compared with a control DNA reference pool containing equal amounts of peripheral blood-derived DNA from 10 random individuals, enabling a subsequent mathematical establishment of relative copy number changes.
Tiling-resolution array-based CGH
In this study, we have used a tiling-resolution genomic microarray consisting of 32 447 overlapping BAC clones selected to cover the entire human reference genome (Ishkanian et al., 2004; Krzywinski et al., 2004), which was prepared as previously described (Veltman et al., 2004; de Vries et al., 2005). In short, genomic target DNAs were isolated from bacterial cultures using an AutogenPrep 960 (Autogen, Holliston, MA, USA) according to the manufacturer's instructions. Subsequently, 
50 ng DNA from each of the clones was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR), then dissolved at an average concentration of 1 µg/µl in 30% DMSO-containing spotting buffer, and spotted onto CMT-ultragaps coated glass slides (Corning) using an Omnigrid 100 arrayer (Genomic Solutions).
Labeling and hybridization
DNA was extracted from umbilical cord derived (newborn) or peripheral blood (parents and individuals from control group) using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's procedure with minor modifications. In brief, DNA was isolated after Proteinase K and RNase treatment. Two washing procedures were performed with washing buffer AW2. DNA was eluted in AE buffer. Subsequent hybridizations were performed basically as described previously (Vissers et al., 2003). In brief, 500 ng of genomic DNA from the umbilical cord blood sample and that of one of the parents was labeled by random-primed labeling with Cy3-dUTP or Cy5-dUTP (Amersham Biosciences), respectively. Labeled DNAs were mixed with 120 µg human Cot-1-DNA (Roche), co-precipitated and re-suspended in hybridization solution (50% formamide; 10% dextran sulfate; 2x sodium saline citrate (SSC); 4% SDS; 10 µg/µl tRNA (Invitrogen). Slides were hybridized for 18 h at 37°C with active re-circulation of hybridization fluid, followed by five wash cycles in 50% formamide, 2x SSC at 45°C and five wash cycles in phosphate buffer at 20°C. Subsequently, slides were dried by centrifugation and scanned on a GenePix Autoloader 4200AL laser scanner (Axon Instruments, Union City, CA, USA). Analysis of the microarray image files was performed with the GenePix Pro 5.0 software package (Axon Instruments), and all data were uploaded into a local database expert system for automated data-normalization and further analysis.
Data processing and statistical analysis
Genomic intervals showing altered test/reference (T/R) fluorescent intensity values, and as such qualifying as candidate regions for putative de novo copy number alterations, were detected by expert eye, with the standard aid of hidden Markov modeling (HMM) as described previously (de Vries et al., 2005). Initial statistical analysis was performed using Fisher's exact test. A statistical significant difference was defined as P < 0.05. All identified genomic copy number alterations were compared with both public and private databases of known disease-unrelated large-scale CNVs (http://projects.tcag.ca/variation/). Gene organization of the regions involved was obtained from public databases (mainly: http://genome.ucsc.edu NCBI genome build 36.1, March 2006).
Pyrosequencing
In order to further evaluate the array-CGH findings, and in an attempt to determine absolute genomic copy numbers for selected genomic intervals in both children as well as their biological parents, pyrosequencing (PSQ) reactions were performed for multiple SNPs residing within these genomic regions. PSQ was selected as the most appropriate technological approach as its dynamic detection-range extends well beyond that offered by other approaches, for instance, MLPA (L.E.L.M. Vissers, personal communication). Interphase fluorescence in situ hybridization (FISH) analysis was considered not to be appropriate as the previously established limited genomic distance of some of the repeat-intervals under investigation was assumed to impair reliable copy number counts due to fluorescent signal-merging. PSQ (for an explanation of the technique, see http://www.pyrosequencing.com) was performed according to the protocol of the manufacturer, with minor modifications (Ruiter et al., 2007). Briefly, ratios of SNP genotypes were determined using a Pyrosequencer PSQ96MA platform (Biotage AB, Uppsala, Sweden). SNP-specific PSQ primer combinations were developed in-house, using dedicated ‘PSQ Assay Design’ software (Isogen Life Science). Primers were ordered from Biolegio BV, Nijmegen, The Netherlands, or Isogen Life Science, IJsselstein, The Netherlands. SNP-specific PSQ primer sequences are available from the authors upon request. PSQ reactions were performed in triplicate.
Copy number estimations
Genomic copy number estimations were mainly based on observed SNP genotype ratios. In addition, the number of copies apparently present within the human reference genome was estimated through BLAT searches at the UCSC Genome Bioinformatics site (http://genome.ucsc.edu/cgi-bin/hgBlat), using sequences (typically a few hundred bases) encompassing the SNP under investigation (for an example see lower panel of Fig. 1, which reveals that SNP RS16875985 appears to be present in seven copies per haploid reference genome). Arbitrary ‘reference copy numbers’ obtained in this way were used as initial offsets to establish ‘best-fit copy numbers’ in ICSI children and their parents. In addition, also average, region-specific T/R values (Fig. 1 upper panel) were taken into consideration.
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Results |
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One year after birth, all parents of the study group were sent detailed questionnaires in order to establish the medical condition of their children. In addition, children born after ICSI with sperm from a PESA procedure went through an extensive medical examination at the age of 2 years in the context of standard national ICSI-PESA guidelines. The mean duration of pregnancy of the study group was 39+3 weeks (range: 36–42+1). Their mean birthweight was 3578 g (range: 2610–4320). One child was part of a twin pregnancy with duration of 36 weeks and a birthweight of 2610 g (the other part twin was not included in the study group, but was also healthy). None of the children had a minor or major malformation, and none was admitted at the hospital in the first year of life.
Array-CGH
Using a HMM algorithm with standard settings, as explained in more detail in the Materials and Methods section, genomic copy number differences with respect to a single parent could easily be identified. The large majority of these changes, however, seemed to represent common variations in copy number (http://projects.tcag.ca/variation/), which are known to be fully compatible with normal Mendelian inheritance. It is therefore reasonable to assume that the large majority of these are unlikely to represent de novo genomic copy number changes.
In a number of instances, ‘same direction copy number changes’ [i.e. simultaneous copy number gain (or loss) with respect to both parents] were identified. There were 10 such genomic intervals in six children detected among the ICSI children (n = 12). All these intervals belonged to a previously defined set of 13 most frequently encountered regions of genomic polymorphism (Redon et al., 2006; White et al., 2007). Scanning of the control group of 30 child–parent trios for similar same direction copy number changes in these intervals revealed six similar events in five children (Table I).
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A representative example of one such interval (targeting chromosome 5q12, family K) is shown in Fig. 1. For this case, the BAC array (upper panel) shows a clear copy number gain of the newborn compared with both the father (plotted in green) and the mother (plotted in red).
Pyrosequencing
On the basis of available HapMap data (http://www.hapmap.org/) SNP-linked PSQ primers were designed and optimized for a total of 14 loci, representing five of the seven genomic intervals listed in Table I. No informative SNPs could be identified for the 0.5 Mb interval on chromosome 22q11. Primer-combinations and optimized PCR-/PSQ-conditions are readily available upon request from the authors. Primary data-processing using standard PSQ96MA software (Biotage AB), yielded reproducible SNP genotype ratio calls for all the SNPs typed (primary experimental data are available upon request). Figure 2 shows a composite figure containing representative examples of the graphical output produced by the PSQ96MA software package (family K).
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Statistical analysis
There were 10 ‘same direction copy number changes’ detected among six ICSI children (total number tested: 12), whereas only six similar genomic regions in five children could be detected within our much larger (n = 30) control group (Table I). The two tailed P-value by Fisher's exact test for these values equals 0.049, so ‘same direction copy number changes’ are significantly more frequent within our panel of ICSI children than among children from our control group. Although the numbers are small, in the ART group (n = 10), all but one were losses, whereas for the control group (n = 6) there was a balance between losses and gains (Table I). Hence, when testing for losses only, a higher level of significance is obtained (P = 0.031).
There were six copy number changes found in four children (out of six) born after ICSI with sperm from a PESA, and four changes in two children (out of six) born after ICSI with ejaculated sperm from fathers suffering extreme OAT. The two-tailed P-value by Fisher's exact test equals 0.567, indicating that there is no significant chance difference for having ‘same direction copy number changes’ between children born after ICSI with sperm from a PESA and children born after ICSI with ejaculated sperm from fathers suffering extreme OAT.
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Discussion |
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As mentioned in the Introduction, large follow-up studies by several independent research groups have revealed a small though statistically significant increase in the incidence of certain birth defects in ICSI children compared with naturally conceived infants (Hansen et al., 2002
, 2005; Zhu et al., 2006). Also a significantly higher rate of de novo chromosomal anomalies has been observed in ICSI-mediated offspring (Bonduelle et al., 2002). These data suggest that ART procedures increase the genetic load. Such an increase, if present, could, besides gross chromosomal changes including CNV of large kilobyte range sequences, also apply to other mutation types such as simple sequence repeat instability, small deletions, duplications, inversions and base pair changes. At the moment, we would not know to what extent an increased genetic load might manifest itself in congenital malformations. The lack of knowledge regarding the primary molecular mechanism(s) underlying the observed ICSI-associated increased rate of birth defects, prompted us to initiate the study described here. We used BAC array mediated CGH (array-CGH) analysis in order to identify possible constitutional de novo DNA copy number changes in ICSI children, which can be detected with the 32k array with a resolving power of around 100 kb. Up to now, the considerable CNV detected with this type of technology at first observation appears to be phenotypically silent, although selective pressures must have been operative (Cooper et al., 2007; Jiang et al., 2007). There were 10 ‘same direction copy number changes’ [i.e. simultaneous copy number gain (or loss) with respect to both parents] found in 6 out of the 12 ICSI children, 9 of which were losses. In an attempt to obtain a more accurate basis for our copy number estimates, we subsequently used PSQ as an independent technology platform to validate our array-CGH findings. PSQ data were obtained for all child–parents trios with copy number changes (except for an apparent de novo copy number change at chromosome 22q11.1, for which no suitable SNPs could be selected) and were fully compatible with our BAC array-based observations. As could be expected, PSQ does not provide the investigator with exact copy number counts. The haplotype ratios obtained through this technique, however, can be very helpful nevertheless, as they provide a kind of mathematical framework (i.e. ‘multiples of ... ’, as explained in the legend to Fig. 2) for copy number estimates. It should be realized, however, that the ratios obtained by this technique can be explained either by a minimal ratio-compatible number of copies or a multiple of this number, thereby always providing the mathematical opportunity for a standard Mendelian segregation fit (assuming that copy numbers are not limited). Moreover, this technique is unsuitable for determining the distribution of these copies over the chromosome pair (which would require more extensive segregation analysis or combined sperm- and oocyte-typing).
Since no information can be obtained regarding the distribution of individual copies over the two autosomes, we decided to use statistics to investigate whether the frequency at which ‘same direction copy number changes’ were encountered in our patient population was significantly different from that found in our reference population. The two-tailed P-value by Fisher's exact test indicated that ‘same direction copy number changes’ appear to be significantly more frequent within our panel of ICSI children than among children from our control group (P = 0.049). This result is more striking when only repeat losses are considered (P = 0.031). No significant difference was found for ‘same direction copy number changes’ in children born after ICSI with sperm from a PESA compared with children born after ICSI with ejaculated sperm from fathers suffering extreme OAT (P = 0.567). Our study group was small, so a larger group is needed to confirm these findings.
A Mendelian explanation of our results would assume appreciable size differences between the two ‘alleles’ from both parents. The reason for ART in these couples was a male infertility problem. Moreover, the association between allele size difference and infertility is at least questionable because of the absence of a theoretical/genetic explanation in the literature. So the de novo occurrence of a CNV, notably losses, in the male germline is the most plausible explanation.
In PESA patients, spermatogenesis is assumed to be normal. However, de Boer et al. (2004) found indications for altered kinetics of meiotic prophase in this class with leptotene and late zygote spermatocytes being overrepresented. This is suggestive for difficulties in initiating and finalizing meiotic synapsis and hence crossing-over (Cromie and Smith, 2007). Recently, a decreased efficiency at meiotic synaptic initiation and finalization has also been found in cases of human azoospermia (Sun et al., 2007).
This observation suggests a link between the increased occurrence of CNV and a disturbed kinetics of first meiotic prophase, likely involving the organization of the axial lateral elements of the synaptonemal complex, as this organization determines which homologous sequences are brought together in early and late recombination nodules (de Boer et al., 2006; Novak et al., 2008).
Impaired progression at the end of zygotene is most prominent at the large heterochromatic blocks that are often different in size between homologues, adjacent to the centromeres of chromosomes 9 and 1 (Codina-Pascual et al., 2006). Recently, it has been found that delayed synapsis in these regions also can show as a recombination effect in trans on chromosome 5 (Sun et al., 2007), where we found CNV at 5q13.2 most frequently.
Alternatively, the origin could be at the elongated spermatid and sperm stages as these, due to contracted chromatin are regarded as devoid of DNA repair, that is subsequently triggered in the zygote. Double strand DNA repair by non homologous end joining and homologous recombination is active in the zygote (Marchetti et al., 2007; Derijck et al., 2008), and a male-female interaction at this level for the generation of paternal Retinoblastoma mutations has been proposed (Kato et al., 2007). In this scenario, stalled replication forks in the paternal pronucleus by compromised homologous recombination repair (Derijck et al., 2008) would lead to the mainly deletions in large scale repeat areas.
An extension of the explanation of our results has recently been offered by CNV analysis of phenotypically concordant and discordant monozygotic twins (Bruder et al., 2008). Somatic mosaicism was shown to be common in peripheral blood-derived DNA, both by a tiling 32K BAC array and by an Illumina system SNP analysis. Hence, the origin of the mutation event, likewise connected with repeat replication stalling, could also be later than the first cleavage division, with later origins expected to contribute less of a CGH effect, if not outweighted by the size of the event.
As far as can be judged by medical examination of the young children of concern here, the genomic changes observed do not have a phenotypic consequence. Because of the small sample size, phenotypic effects were not expected. However, as repeat areas are not by definition genetic deserts (see for instance Fig. 1) and genes in these and comparable areas have been linked to phenotypic effects, dosage effects could be at the basis of congenital malformation. For instance, genomic alterations affecting SMN1 (see Fig. 1) are linked to the development of spinal muscular atrophy (http://www.genecards.org/).
Although executed at a small scale, our search revealed a relative abundance of ‘same direction copy number changes’ in an ART setting, increased by a factor of 5 relative to the control population. Our estimate likely is an underestimate as the two alleles are pooled per individual. Hence, meiotic segregation from one parent can obscure a de novo event from the other parent (for a de novo deletion this would mean the inheritance of a longer repeat array from the other parent).
In conclusion, the recently discovered genome mobility by CNV in the human likely is enhanced in an ART setting, a result that should warrant more detailed investigation into especially the mechanisms and germ cell stages involved.
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Funding |
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Foundation Clinical and Experimental Investigations Department of Obstetrics and Gynecology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
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Acknowledgements |
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The authors thank: the families for their willingness to participate in this study, Nicole Wellens (Isogen Life Science) for outstanding customer support and invaluable help with the pyrosequencing study design, Marga Schepens for excellent technical assistance, and Joris Veltman, Bart Kiemeney and Jan Hendriks for helpful suggestions and stimulating discussions.
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Submitted on April 23, 2008; resubmitted on June 20, 2008; accepted on July 22, 2008.
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