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<?xml version="1.0" ?> <tei> <teiHeader> <fileDesc xml:id="_1755-8166-2-3"/> </teiHeader> <text xml:lang="en"> <note place="headnote">BioMed Central <lb/></note> <page> Page 1 of 11 <lb/></page> <note place="footnote"> (page number not for citation purposes) <lb/></note> <front> Molecular Cytogenetics <lb/> Open Access <lb/> Case report <lb/> Meiotic and mitotic behaviour of a ring/deleted chromosome 22 in <lb/>human embryos determined by preimplantation genetic diagnosis <lb/>for a maternal carrier <lb/> Anna Mantzouratou* 1,2 , Anastasia Mania 1 , Marianna Apergi 1 , Sarah Laver 2 , <lb/>Paul Serhal 2 and JDA Delhanty 1 <lb/> Address: 1 UCL Centre for PGD, Institute for Women's Health, University College London, 86-96 Chenies Mews, London, WC1E-6HX, UK, 2 The <lb/>Assisted Conception Unit, University College Hospital, Eastman Dental Hospital, Gray's Inn Road, London, WC1X 8LD, UK and <lb/>Email: Anna Mantzouratou* -a.mantzouratou@ucl.ac.uk; Anastasia Mania -anastasia.mania@ucl.ac.uk; <lb/>Marianna Apergi -m_apergi@yahoo.co.uk; Sarah Laver -Sarah.Laver@uclh.nhs.uk; Paul Serhal -paul.serhal@uclh.nhs.uk; <lb/>JDA Delhanty -j.delhanty@ucl.ac.uk <lb/>* Corresponding author <lb/> Abstract <lb/> Background: Ring chromosomes are normally associated with developmental anomalies and are <lb/>rarely inherited. An exception to this rule is provided by deletion/ring cases. We were provided <lb/>with a unique opportunity to investigate the meiotic segregation at oogenesis in a woman who is a <lb/>carrier of a deleted/ring 22 chromosome. The couple requested preimplantation genetic diagnosis <lb/>(PGD) following the birth of a son with a mosaic karyotype. <lb/>The couple underwent two cycles of PGD. Studies were performed on lymphocytes, single <lb/>embryonic cells removed from 3 day-old embryos and un-transferred embryos. Analysis was <lb/>carried out using fluorescence in situ hybridisation (FISH) with specific probe sets in two rounds <lb/>of hybridization. <lb/> Results: In total, 12 embryos were biopsied, and follow up information was obtained for 10 <lb/>embryos. No embryos were completely normal or balanced for chromosome 22 by day 5. There <lb/>was only one embryo diagnosed as balanced of 12 biopsied but that accumulated postzygotic errors <lb/>by day 5. Three oocytes apparently had a balanced chromosome 22 complement but all had the <lb/>deleted and the ring 22 and not the intact chromosome 22. After fertilisation all the embryos <lb/>accumulated postzygotic errors for chromosome 22. <lb/> Conclusion: The study of the preimplantation embryos in this case provided a rare and significant <lb/>chance to study and understand the phenomena associated with this unusual type of anomaly during <lb/>meiosis and in the earliest stages of development. It is the first reported PGD attempt for a ring <lb/>chromosome abnormality. <lb/></front> <body>Background <lb/> In humans, ring chromosomes are normally associated <lb/>with developmental anomalies and are rarely inherited. <lb/>An exception to this rule is provided by del/ring cases <lb/>where euchromatic material carried by the ring has been <lb/>derived by an interstitial deletion, with one of the breaks <lb/></body> <front>Published: 23 January 2009 <lb/> Molecular Cytogenetics 2009, 2:3 doi:10.1186/1755-8166-2-3 <lb/>Received: 3 December 2008 <lb/>Accepted: 23 January 2009 <lb/>This article is available from: http://www.molecularcytogenetics.org/content/2/1/3 <lb/> © 2009 Mantzouratou et al; licensee BioMed Central Ltd. <lb/>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), <lb/>which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <lb/> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></front> <page>Page 2 of 11 <lb/></page> <note place="footnote">(page number not for citation purposes) <lb/></note> <body>occurring through the centromere. Ring/del cases can be <lb/>considered to form a special subgroup among the small <lb/>supernumerary marker chromosomes (sSMC); these are <lb/>additional, abnormal chromosomes the origin of which <lb/>cannot usually be determined by conventional tech-<lb/>niques. If the additional genetic material is of euchro-<lb/>matic origin the sSMC may be associated with <lb/>developmental anomalies, but in the case of the ring/del <lb/>situation the additional material is compensated for by <lb/>the deletion and the phenotype is normal. <lb/>Several examples of the ring/deleted type of anomaly are <lb/>known from the literature [1-10]. The rearrangement cre-<lb/>ates a balanced carrier status, with the potential to pro-<lb/>duce abnormal offspring with a variety of unbalanced <lb/>karyotypes, either duplicated or deleted for the region <lb/>involved [1,4,6,7]. Prenatal diagnosis may be offered to <lb/>known del/ring carriers but risk calculations will be diffi-<lb/>cult since the meiotic behaviour of this type of anomaly is <lb/>unknown in humans. <lb/>We were provided with a unique opportunity to investi-<lb/>gate the meiotic segregation at oogenesis in a woman who <lb/>is a carrier of a del/ring 22 chromosome. The couple <lb/>requested preimplantation genetic diagnosis (PGD) fol-<lb/>lowing the birth of a son with a mosaic karyotype. In his <lb/>lymphocytes, one cell line had a copy of the ring 22 chro-<lb/>mosome in addition to the normal 46,XY complement <lb/>while in other cells the ring had been lost. A subsequent <lb/>female pregnancy with the same karyotype was termi-<lb/>nated. The first pregnancy had followed a period of infer-<lb/>tility. <lb/>For PGD, the couple involved has to undergo in vitro fer-<lb/>tilization (IVF) to enable the simultaneous testing of sev-<lb/>eral embryos. One or two cells from each embryo is <lb/>removed on day 3 of development and tested for the par-<lb/>ticular chromosome(s) involved to allow selection of <lb/>those that are unaffected [11]. After two PGD treatment <lb/>cycles and two natural pregnancies, information was <lb/>available on 12 meioses, none of which produced an <lb/>oocyte with a single intact chromosome 22. Three meioses <lb/>resulted in balanced oocytes carrying both the deleted <lb/>chromosome 22 and the ring, as in the mother. Due to the <lb/>known instability of small ring chromosomes, as evi-<lb/>denced both in this family and others in the literature <lb/>[7,12,13] this situation created a dilemma when detected <lb/>at embryo biopsy. The follow up studies carried out on the <lb/>embryos that were not transferred after diagnosis pro-<lb/>vided an opportunity to monitor the mitotic behaviour of <lb/>the ring 22 chromosome. <lb/> Results <lb/> When considering the appropriate probes for this unusual <lb/>abnormality it was decided that the balanced carriers of <lb/>the maternal rearrangement needed to be detected and <lb/>distinguished from embryos that had two normal copies <lb/>of chromosome 22 due to the high risk of instability lead-<lb/>ing to mosaicism that is associated with the ring/del type <lb/>of abnormality [7,12,13]. Cytogenetic workup in parental <lb/>lymphocytes with commercially available probes for chro-<lb/>mosome 22 showed that the centromere of chromosome <lb/>22 was split between both derivative chromosomes 22 <lb/>(the deleted chromosome and the ring) in the mother. <lb/>Consequently, two rounds of FISH were used in order to <lb/>detect all the unbalanced and balanced carriers in the <lb/>resulting embryos. The combined FISH probe efficiency <lb/>on control lymphocytes was 90% and on patient lym-<lb/>phocytes was 95%. Figures 1 and 2 show the FISH results <lb/>on control and patient lymphocytes for both metaphase <lb/>and interphase nuclei. Figure 3 shows an example of the <lb/>FISH results on embryonic nuclei from biopsied and <lb/>untransferred embryos which are all in the interphase <lb/>stage. <lb/>The couple underwent two cycles of preimplantation <lb/>genetic diagnosis for the rare ring/del chromosome <lb/>abnormality. There was no embryo transfer in either cycle <lb/>due to all the embryos being either affected with a chro-<lb/>mosome 22 imbalance or balanced carriers of the mater-<lb/>nal chromosomal abnormality. Table 1 (Additonal File 1) <lb/>summarises the results of the first PGD cycle. In brief, 7 <lb/>oocytes were collected and 6 of them were fertilised by <lb/>IVF. Five embryos were biopsied on day 3 and two cells <lb/>were taken from all embryos. Unfortunately, no embryos <lb/>were available for transfer. An embryo that was a balanced <lb/>carrier of the ring/del chromosome was found but due to <lb/>the likely instability of the ring 22 during cell division (as <lb/>evidenced by cytogenetic analysis of tissues from the cou-<lb/>ple's two natural pregnancies), the couple decided not to <lb/>have it transferred. All biopsied embryos were spread on <lb/>slides on day 5 for full follow up analysis. Four out five <lb/>embryos grew well and had reached the expected blasto-<lb/>cyst stage. Results were obtained on follow up for four out <lb/>of the five embryos (Table 2 -Additional File 1). In three <lb/>embryos the meiotic imbalance detected at biopsy was <lb/>confirmed but they were also aneuploid mosaics due to <lb/>additional post-zygotic errors. Embryo number 2 that was <lb/>diagnosed as a balanced ring/del carrier on day 3 had <lb/>become mosaic abnormal by day 5 with loss of the ring in <lb/>50% of the cells. The meiotic segregation patterns of the <lb/>recombinant and normal 22 chromosomes in the oocytes <lb/>were deduced according to the biopsy results and subse-<lb/>quent follow up data. The combined evidence suggests <lb/>that one of the five oocytes started with the ring and <lb/>deleted 22, two started with only the deleted chromosome <lb/>22 present and the remaining two started with the normal <lb/>22 and the deleted 22 present (Table 2 -Additional File 1). <lb/> <note place="headnote">Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 3 of 11 <lb/></page> <note place="footnote">(page number not for citation purposes) <lb/></note> Table 3 (Additional File 1) summarises the results of the <lb/>second PGD cycle. During this cycle, 10 oocytes were col-<lb/>lected, eight of which were normally fertilised by IVF. <lb/>Seven embryos were biopsied, two cells were taken from <lb/>three embryos and one cell was taken from 4 embryos <lb/>with fewer than six cells. Table 4 (Additional File 1)shows <lb/>the biopsy and follow up results for this cycle. No <lb/>embryos normal or balanced for chromosome 22 were <lb/>found at diagnosis in this cycle. Embryo 4 had the ring <lb/>and deleted 22 present but the normal 22 (presumably <lb/>paternal in origin) was lost in the biopsied cell. <lb/>All embryos were arrested in development at the 3–10 cell <lb/>stage by day 5. Follow up results were obtained for six un-<lb/> Hybridization of diagnostic probe sets to lymphocyte nuclei of the father <lb/> Figure 1 <lb/>Hybridization of diagnostic probe sets to lymphocyte nuclei of the father. In the first round, the Di George dual <lb/>band probe set is used (22q11.2 orange/22q13.3 green). In the second round the probes to detect the centromere 14/22 red <lb/>and 14qtel in green are used. <lb/> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/>Page 4 of 11 <lb/> (page number not for citation purposes) <lb/> transferred embryos. Four embryos were fully chaotic i.e. <lb/>with random abnormalities varying from cell to cell with <lb/>no discernable mechanism. Combined diagnostic and fol-<lb/>low up results for Embryo 5 suggest that this too originally <lb/>had both the ring and deleted chromosome 22 present, <lb/>but chaotic cell divisions led to the loss of either the ring <lb/>or the deleted 22 in different cells. The partial monosomy <lb/>22 found at biopsy in Embryo 1 was confirmed but it was <lb/>an aneuploid/chaotic mosaic by day 5. Embryo 3 <lb/>appeared to be haploid with a single intact copy of chro-<lb/>mosome 22 and a single copy of chromosome 14. The <lb/>remaining embryo (number 6) had partial monosomy 22 <lb/>but that was based on only one cell and could not be con-<lb/>firmed. The follow up results helped in the deduction of <lb/> Hybridization of diagnostic probe sets to lymphocytes of the carrier mother <lb/> Figure 2 <lb/>Hybridization of diagnostic probe sets to lymphocytes of the carrier mother. In the first round, the Di George dual <lb/>band probe set is used (22q11.2 orange/22q13.3 green). In the second round the probes to detect the centromere 14/22 red <lb/>and 14qtel in green are used. <lb/> <note place="headnote"> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 5 of 11 <lb/></page> <note place="footnote"> (page number not for citation purposes) <lb/></note> the meiotic segregation of chromosome 22 in the oocytes. <lb/>It was deduced that of five oocytes two had the ring and <lb/>the deleted chromosome 22 present, one oocyte had the <lb/>deleted and inact chromosome 22 and another two had <lb/>the ring chromosome 22 only; the latter types would lead <lb/>to partial trisomy or monosomy 22 after fertilisation. In <lb/>degenerate embryo number 6 there was insufficient relia-<lb/>ble information to be able to determine the chromosomal <lb/>complement of the oocyte (Table 4 -Additional File 1). <lb/>In total, 12 embryos were biopsied, and follow up infor-<lb/>mation was obtained for 10 embryos. No embryos were <lb/>completely normal or balanced for chromosome 22 by <lb/>day 5. There was only one embryo diagnosed as balanced <lb/>out of 12 biopsied and in that embryo by day 5 postzy-<lb/>gotic errors lead to a mosaic karyotype with half the cells <lb/>having lost the ring chromosome. According to the follow <lb/>up studies, in all, three oocytes apparently had a balanced <lb/>chromosome 22 complement but all had the deletion and <lb/>the ring 22 and not the intact chromosome 22. After ferti-<lb/>lisation all three of the embryos accumulated postzygotic <lb/>errors for chromosome 22 with the end result being either <lb/>diploid/aneuploid mosaic or chaotic mosaic. Embryo 3 <lb/>from cycle 2 appeared to be haploid with a single intact <lb/>copy of chromosome 22; re-probing for chromosomes X, <lb/>Y and 18 confirmed haploidy but with a single X chromo-<lb/>some so that the parental origin could not be determined. <lb/>The rest of the oocytes are thought to have been the unbal-<lb/>anced products of meiosis. Post-zygotic errors in the <lb/>resulting embryos were wide ranging and very frequent in <lb/>almost all the embryos. Table 5 (Additional File 1) sum-<lb/>marises the theoretical chromosomal complement in <lb/>oocytes collected during the PGD cycles and in the two <lb/>natural pregnancies. Both natural ongoing pregnancies <lb/>resulted from oocytes that had an extra ring chromosome <lb/>(24,X,+r22) but none of the embryos from PGD cycles <lb/>presented with this combination. <lb/> Discussion <lb/> A 37 year old female carrier of a rare chromosome rear-<lb/>rangement was referred for PGD. She was a balanced car-<lb/>rier of a deleted 22 and a complementary ring <lb/>chromosome 22. This is the first report of PGD for this <lb/>kind of abnormality as it is extremely rare. Two cycles of <lb/>PGD were carried out using FISH with case specific <lb/>probes. The strategy devised for this couple was effective <lb/>since the balanced carriers of the rearrangement and all <lb/>unbalanced forms were identified. The position of the <lb/>breakpoint within the centromere of chromosome 22 <lb/>meant that by using the centromeric probe for chromo-<lb/>somes 14/22 in conjunction with the subtelomere probe <lb/>of 14q in the second round it was possible to identify the <lb/>number of centromeric signals for chromosome 22 and <lb/>hence the derivative chromosomes. Unfortunately there <lb/>were no embryos suitable for transfer in either cycle since <lb/>none had two intact chromosomes 22. All untransferred <lb/>embryos were fully analysed providing a rare glimpse of <lb/> Results of FISH analysis on embryonic nuclei with probes for the detection of the intact chromosome 22 and the ring/deleted <lb/>22 chromosome during PGD <lb/> Figure 3 <lb/>Results of FISH analysis on embryonic nuclei with probes for the detection of the intact chromosome 22 and <lb/>the ring/deleted 22 chromosome during PGD. A. First round FISH result with the Di George dual band probe set <lb/>(22q11.2 orange/22q13.3 green).on a single blastomere showing loss of ring chromosome 22 detected by the orange probe. B. <lb/>Normal FISH signals for chromosome 22 with the Di George dual band probe set (22q11.2 orange/22q13.3 green)., the <lb/>embryo could have two intact chromosomes 22 or be a carrier. C. Second round FISH result with probes Cep14/22 (red) and <lb/>14qtel (green) on the same cell as in B, shows a balanced carrier of the ring and deleted 22. <lb/> <note place="headnote">Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 6 of 11 <lb/></page> <note place="footnote">(page number not for citation purposes) <lb/></note> the behavior of the derivative chromosomes 22 during <lb/>oogenesis and preimplantation development. <lb/>Considering the meiotic behaviour of the ring chromo-<lb/>some, all possible meiotic segregation patterns were seen <lb/>and there does not appear to be a preferential segregation <lb/>mode. Assuming that the intact chromosome 22, together <lb/>with the deleted and the ring chromosome 22 were able <lb/>to form a trivalent during prophase of meiosis I then there <lb/>are three possible modes of segregation, only one produc-<lb/>ing balanced gametes (see Figure 4). Unbalanced products <lb/>of the other two modes were all seen either in the natural <lb/>pregnancies or in the embryos generated by IVF for PGD. <lb/>In the case of the three embryos with presumed balanced <lb/>carrier status it is assumed that the intact chromosome 22 <lb/>passed to the first polar body. The evidence suggests that <lb/>there is in fact random segregation in this case, presuma-<lb/>bly because normal pairing and recombination is dis-<lb/>rupted. The reduced size of the centromeric sequences that <lb/>exist in both the del(22) and the r(22) might also affect <lb/>attachment to the meiotic spindle [14,15]. <lb/>In one of her seminal contributions Barbara McClintock <lb/>describes the mechanism leading to the formation of ring/ <lb/>deleted chromosomes in maize and the aberrant mitotic <lb/>behaviour leading to 'variable mutant characteristics' [16]. <lb/>This mechanism, a break within the centromere together <lb/>with a break in either the long or the short arm, creating a <lb/>small ring, has been called "centromere misdivision" [17]; <lb/>these authors propose that this be referred to as "the <lb/>McClintock mechanism". McClintock also describes <lb/>pachytene configurations in microsporocytes, showing <lb/>that although the normal, deleted and ring chromosomes <lb/>may synapse, the ring is also seen with the centromeric <lb/>region attached to a non-homologous bivalent. In the <lb/>human situation with the ring chromosome 22, this could <lb/>well be the chromosome 14 bivalent, since there is sub-<lb/>stantial centromeric homology between the two chromo-<lb/>somes. A rare case of a ring Y chromosome transmitted <lb/>from a father to his son, who was affected by Klinefelter <lb/>syndrome of paternal origin, also illustrates the effect of <lb/>the ring on normal segregation of the X and Y chromo-<lb/>somes [18]. Additionally, several examples of maternal <lb/> Segregation patterns in meiosis I of oogenesis in a female carrier of del 22/ring 22 chromosomes <lb/> Figure 4 <lb/>Segregation patterns in meiosis I of oogenesis in a female carrier of del 22/ring 22 chromosomes. <lb/> <note place="headnote"> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 7 of 11 <lb/></page> <note place="footnote"> (page number not for citation purposes) <lb/></note> and paternal transmision of similar sSMC can be found in <lb/>the sSMC homepage http://www.med.uni-jena.de/fish/ <lb/>sSMC/00START.htm. In this case, FISH analysis of sperm <lb/>from father and son showed significantly higher frequen-<lb/>cies of diploidy and various disomies compared with con-<lb/>trol samples, suggesting that the reduced pairing efficiency <lb/>of the ring Y chromosome disrupted the meiotic process <lb/>generally. This is less likely to occur in the human female, <lb/>since meiotic cell cycle checkpoints are less stringent than <lb/>in the male [19]. <lb/>Postzygotic errors were also widespread in all pre-implan-<lb/>tation embryos studied in this case resulting in a high <lb/>degree of mosaicism. The two natural conceptions also <lb/>showed mosaicism. The instability of ring chromosomes <lb/>is well documented in other studies both in prenatal sam-<lb/>ples and liveborn offspring[7,20-22]. The origin of this <lb/>instability is partly attributed to the nature of ring chro-<lb/>mosomes and their difficulty in undergoing mitotic divi-<lb/>sion, with a tendency to form interlocking rings, leading <lb/>to anaphase lag and chromosome loss [16]. In the preim-<lb/>plantation embryos and natural conceptions of the case <lb/>studied, however, a varying degree of instability is <lb/>observed in 100% of their embryonic and fetal offspring. <lb/>Multiple cell lines can be seen in the preimplantation <lb/>embryos due to loss of the smaller derivative chromo-<lb/>somes 22, or the intact chromosome, and due to chaotic <lb/>cell divisions. The initial meiotic error and the instability <lb/>of the r(22) and the del(22) is in addition to the frequent <lb/>unbalanced mitotic divisions common in the case of pre-<lb/>implantation embryos. It is likely that the extreme chaotic <lb/>mosaisism seen in many embryos in this case is related to <lb/>the sub-fertility of the couple concerned since this type of <lb/>mosaisism, although seen to be widespread during preim-<lb/>plantation development [23] is substantially increased in <lb/>couples with repeated implantation failure [24]. <lb/>In this case the ring chromosome is very stable in the <lb/>mother as she is phenotypically normal and has the r(22) <lb/>in all metaphases and interphases studied in her lym-<lb/>phocytes. The reasons for this are not well understood. <lb/>One possible explanation is that the centromeric and tel-<lb/>omeric regions required for normal cell division are still <lb/>intact in the mother while in her embryos these regions <lb/>may be missing or significantly shortened and their func-<lb/>tionality reduced. Interestingly, the only well developed <lb/>carrier embryo that appeared to have the same chromo-<lb/>somes as the mother (Embryo 2 in the first cycle) lost the <lb/>ring in half of the cells by the fifth day of development, <lb/>creating a mosaic with a balanced and a partially mono-<lb/>somic cell line. This error must have happened very early <lb/>on in development possibly by the 8 cell stage or earlier, <lb/>when the cell cycle checkpoints are not thought to be fully <lb/>functional [25,26]. The stability of the ring in the mother <lb/>could also mean that after division any abnormal cell <lb/>lines are not viable and die therefore leaving only the bal-<lb/>anced cell lines present. This cannot be proven however <lb/>and the fact that the offspring in this case was mosaic and <lb/>carried viable trisomic cells lines as well as the size of the <lb/>small size of the imbalance (therefore viable in the tri-<lb/>somic or monosomic state) implies that such unbalanced <lb/>cell lines might not perish. <lb/>Although the natural pregnancies of this couple both <lb/>included partial trisomy 22 with mosaicism, it is expected <lb/>that any of their embryos created by IVF could have pro-<lb/>duced viable unbalanced pregnancies either with partial <lb/>trisomy or partial monosomy due to the small size of the <lb/>chromosome involved. Chromosome imbalance of the <lb/>ring 22 would be 0.6% of HAL and well within the limits <lb/>of viability in the monosomic or in the trisomic state in <lb/>the embryos [27]. <lb/>The decision of the couple not to have the good quality <lb/>balanced carrier embryo from cycle 1 transferred appears <lb/>to be a valid one since the derivative chromosomes appear <lb/>to be highly unstable during mitotic divisions and could <lb/>produce varying abnormal phenotypes. Unfortunately, <lb/>counselling couples with similar chromosomal problems <lb/>is not precise. The variability of the breakpoints and the <lb/>rare nature of these rearrangements as well as mosaicism <lb/>and the variable phenotypes that would be produced <lb/>make the task almost impossible. A similar reported case <lb/>of <lb/>a <lb/>maternal <lb/>carrier <lb/>with <lb/>a <lb/>karyotype <lb/>47,XX,del(22)(q11q11.2),+r(22)(q10q11.2) also con-<lb/>cluded that reproductive risks were high due to the viabil-<lb/>ity of conceptions with the trisomic or monosomic states <lb/>involving the ring and the deleted 22 chromosomes [28]. <lb/>Considering all the above the couple concerned in this <lb/>case presents with a poor prognosis in terms of producing <lb/>a karyotypically normal child when all the evidence from <lb/>the embryos and the previous pregnancies is taken into <lb/>account. Although PGD did not produce a normal preg-<lb/>nancy in this case it has helped give the couple some <lb/>answers concerning the nature of the reproductive diffi-<lb/>culties they have encountered. PGD for this type of abnor-<lb/>mality is a viable option providing that the couple is <lb/>counselled that there may be no embryos suitable for <lb/>transfer. The study of the preimplantation embryos in this <lb/>case provided a rare and significant chance to study and <lb/>understand the phenomena associated with this unusual <lb/>type of anomaly during meiosis and in the earliest stages <lb/>of development. <lb/> Materials and methods <lb/> The couple was referred for PGD after the birth of a child <lb/>with an abnormal karyotype initially described as <lb/>47,XY,+r(22)(p11.2q11.2)/46,XY. The child had a <lb/>number of clinical features including bilateral iris colo-<lb/> <note place="headnote"> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 8 of 11 <lb/></page> <note place="footnote"> (page number not for citation purposes) <lb/></note> boma, retinal coloboma, mild hypospadias and global <lb/>developmental delay. After karyotypic analysis of the par-<lb/>ents it was found that the female partner was a carrier of a <lb/>balanced chromosomal rearrangement involving a ring <lb/>chromosome <lb/>22 <lb/>with <lb/>karyotype <lb/>47,XX, <lb/>del(22)(p10q12),+r(22)(q10q12). The ring is present in <lb/>all her lymphocytes and is stable unlike the situation in <lb/>her son where in some lymphocytes the supernumerary <lb/>ring has doubled in size while in others the ring has been <lb/>lost creating a normal chromosomal complement. Subse-<lb/>quently a second natural pregnancy with an abnormal <lb/>karyotype 47,XX,+r(22)(q10.q12)[21]/46,XX[3] was ter-<lb/>minated after chorionic villus sampling. <lb/>The couple underwent two cycles of PGD. The age of the <lb/>female partner was 37 at the start of her first PGD cycle. <lb/>Studies were performed on lymphocytes, single embry-<lb/>onic cells (blastomeres) removed from 3 day-old embryos <lb/>and un-transferred embryos. Analysis was carried out <lb/>using fluorescence in situ hybridisation (FISH) with spe-<lb/>cific probe sets in two rounds of hybridization. Treatment <lb/>and research on embryos from this couple was carried out <lb/>under licences from Human Fertilisation and Embryology <lb/>Authority (HFEA) of the UK. Informed written consent <lb/>was obtained for all procedures. <lb/> Lymphocyte culture and counts <lb/> Karyotyping on all the family members had been per-<lb/>formed by a clinical cytogenetics laboratory prior to the <lb/>onset of treatment. For preparative FISH studies prior to <lb/>PGD lymphocyte cultures from both parents were carried <lb/>out by standard methods. Standard methods of process-<lb/>ing and slide preparation for FISH experiments were then <lb/>used [11]. The efficiency of the FISH probe combination <lb/> Ideogram and probe strategy to detect the ring 22/del 22 chromosome in embryonic nuclei <lb/> Figure 5 <lb/>Ideogram and probe strategy to detect the ring 22/del 22 chromosome in embryonic nuclei. First round hybridi-<lb/>zation: DiGeorge-dual band probes (22q11.2 orange/22q13.3 green). Second round hybridization: Centromere 14/22 red and <lb/>14qtel in green (not shown). <lb/> <note place="headnote">Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 9 of 11 <lb/></page> <note place="footnote">(page number not for citation purposes) <lb/></note> was calculated by counting the number of correct signals <lb/>in 100–200 interphase nuclei from each sample. <lb/> IVF and stimulation protocol, embryos biopsy, blastomere <lb/>and embryo spreading <lb/> Ultrasound guided vaginal oocyte collection was per-<lb/>formed at 37 hours post hCG injection. IVF was per-<lb/>formed at 40 and 41 hours post hCG respectively and was <lb/>dependent on semen parameters and past fertilisation <lb/>rates. Fertilisation was evaluated at 18–20 hours post <lb/>insemination. Embryos were cultured in IVF medium <lb/>(GIII series, Vitrolife, UK). On day 3, embryos that had <lb/>reached at least the four cell stage were biopsied in Ca2+ -<lb/>Mg2+ free biopsy medium (G-PGD, Vitrolife, UK). Two <lb/>cells were removed from embryos consisting of six or <lb/>more cells. Biopsied blastomeres were spread using the <lb/>method described by Harper et al [29]. Cells were washed <lb/>in PBS and transferred to poly-L-lysine slides in spreading <lb/>solution (0.01N HCl, 0.1% Tween 20) which was gently <lb/>agitated until lysis occurred and the nuclei were clear of <lb/>cytoplasm. The co-ordinates of the location of the nuclei <lb/>were noted using an England Finder. The same technique <lb/>was used for whole embryos. <lb/> Fluorescence In Situ Hybridisation <lb/> The combination of FISH probes used in this diagnosis <lb/>was selected according to the breakpoints, firstly to detect <lb/>the unbalanced products of the female meiosis for chro-<lb/>mosome 22 and secondly to distinguish the balanced car-<lb/>rier embryos of the derivative chromosome 22 plus the <lb/>ring chromosome from those with two intact copies of <lb/>chromosome 22. For this purpose FISH probes were <lb/>selected hybridizing to five different sites in two rounds of <lb/>hybridisation. <lb/>In the first round the dual band LSI DiGeorge/VCFS <lb/>region [Spectrum Orange (22q11.2), Spectrum Green <lb/>(22q13.3)] (Abbott, UK) was used. In the second round <lb/>the following probes were used: the centromeric probe for <lb/>chromosome 14/22 in red (Kreatech, UK) in combination <lb/>with the probe for the subtelomere of chromosome 14q <lb/>in green Kreatech, UK). Figure 5 shows the karyotype of <lb/>the carrier parent and the probe strategy used. The detec-<lb/>tion of carriers was possible because the chromosome 14/ <lb/>22 centromeric probe hybridises to both the deleted chro-<lb/>mosome 22 and the ring 22, indicating that one of the <lb/>breakpoints divides the centromeric alpha satellite <lb/>sequences. However, this probe also hybridises to the cen-<lb/> Expected FISH signals in embryonic nuclei of balanced carriers and normal, non carriers, of the ring/deleted chromosome 22 <lb/> Figure 6 <lb/>Expected FISH signals in embryonic nuclei of balanced carriers and normal, non carriers, of the ring/deleted <lb/>chromosome 22. No difference in the number of FISH signals can be detected in the first round. While in the second round <lb/>four equal sized red signals are observed for the non-carrier, the carrier presents five signals three of which are of equal size <lb/>and two that are smaller. The latter combination denotes the splitting of one of the signals for chromosome 22 and thus the <lb/>existence of the deleted and ring chromosomes. <lb/> <note place="headnote"> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/></note> <page>Page 10 of 11 <lb/></page> <note place="footnote"> (page number not for citation purposes) <lb/></note> tromere of chromosome 14 as well as 22 thus the subtelo-<lb/>meric probe for chromosome 14 had to be used in order <lb/>to exclude chromosome 14 from the scoring of the chro-<lb/>mosome 22 centromeres. Figure 6 shows the expected sig-<lb/>nals in embryonic nuclei in diploid carriers and non-<lb/>carriers of the ring 22. <lb/>FISH experiments were undertaken before the PGD treat-<lb/>ment in order to test and optimise conditions for all the <lb/>probe combinations in this study. FISH for all probes was <lb/>carried out using the manufacturer's instruction with <lb/>minor modifications. The slides were examined under an <lb/>epifluorescence Olympus microscope (Olympus BX 40) <lb/>fitted with a Photometrics cooled CCD camera utilising <lb/>Smartcapture software (Digital Scientific, UK). DAPI <lb/>stained nuclei were located using the blue filter. Using dif-<lb/>ferent colour filters the scoring of signals for each of the <lb/>probes to the nuclei on the slides was possible with a good <lb/>degree of accuracy. All scoring decisions were made <lb/>directly by viewing signals under the microscope and ver-<lb/>ified by at least two observers. Scoring criteria were <lb/>applied as described in Mantzouratou et al [24]. <lb/> </body> <back> <div type="annex">Competing interests <lb/> The authors declare that they have no competing interests. <lb/></div> <div type="annex">Authors' contributions <lb/> AM: Main author, involved in preliminary work and all <lb/>data collection and analysis <lb/>MA: Data collection, involved in preliminary lymphocyte <lb/>investigation <lb/>A Mania: Involved in preliminary work and biopsy data <lb/>collection <lb/>SL: Main embryologist in the PGD case <lb/>PS: Main physician in the case <lb/>JD: PGD director, involved in all aspects of this case and <lb/>editing the manuscript. <lb/> Consent <lb/> Written informed consent was obtained from the patient <lb/>for publication of this case report and accompanying <lb/>images. A copy of the written consent is available for <lb/>review by the Editor-in-Chief of this journal. <lb/></div> <div type="annex">Additional material <lb/></div> <div type="acknowledgement">Acknowledgements <lb/> We wish to thank all other members of the UCL Centre for PGD and of <lb/>the Assisted Conception Unit, UCH, for their continuing help and support. <lb/></div> <listBibl> References <lb/> 1. <lb/>Fryns JP, Kleczkowska A, Limbos C, Vandecasseye W, Van den BH: <lb/> Centric fission of chromosome 7 with 47,XX,del(7)(pter----<lb/>cen::q21----qter)+cen fr karyotype in a mother and proximal <lb/>7q deletion in two malformed newborns. 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Fickelscher I, Starke H, Schulze E, Ernst G, Kosyakova N, Mkrtchyan <lb/>H, MacDermont K, Sebire N, Liehr T: A further case with a small <lb/>supernumerary marker chromosome (sSMC) derived from <lb/>chromosome 1 – evidence for high variability in mosaicism in <lb/>different tissues of sSMC carriers. <lb/> Prenat Diagn 2007, <lb/> 27(8):783-5. <lb/></listBibl> <div type="annex"> Additional file 1 <lb/> Manuscript tables. This file includes all tables of this manuscript. <lb/> Click here for file <lb/>[http://www.biomedcentral.com/content/supplementary/1755-<lb/>8166-2-3-S1.doc] <lb/></div> <note place="footnote"> Publish with Bio Med Central and every <lb/>scientist can read your work free of charge <lb/> "BioMed Central will be the most significant development for <lb/>disseminating the results of biomedical researc h in our lifetime." <lb/> Sir Paul Nurse, Cancer Research UK <lb/> Your research papers will be: <lb/> available free of charge to the entire biomedical community <lb/>peer reviewed and published immediately upon acceptance <lb/>cited in PubMed and archived on PubMed Central <lb/>yours — you keep the copyright <lb/> Submit your manuscript here: <lb/> http://www.biomedcentral.com/info/publishing_adv.asp <lb/> BioMedcentral <lb/></note> <listBibl> Molecular Cytogenetics 2009, 2:3 <lb/> http://www.molecularcytogenetics.org/content/2/1/3 <lb/>Page 11 of 11 <lb/> (page number not for citation purposes) <lb/> 14. 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Hum Reprod 2005, 20(2):462-468. <lb/> 19. LeMaire-Adkins R, Radke K, Hunt PA: Lack of checkpoint control <lb/>at the metaphase/anaphase transition: a mechanism of mei-<lb/>otic nondisjunction in mammalian females. J Cell Biol 1997, <lb/> 139(7):1611-9. <lb/> 20. Jeffries AR, Curran S, Elmslie F, Sharma A, Wenger S, Hummel M, <lb/>Powell J: Molecular and phenotypic characterization of ring <lb/>chromosome 22. Am J Med Genet A 2005, 137(2):139-47. <lb/> 21. Liehr T, Mrasek K, Weise A, Dufke A, Rodríguez L, Martínez Guardia <lb/>N, Sanchís A, Vermeesch JR, Ramel C, Polityko A, Haas OA, Ander-<lb/>son J, Claussen U, von Eggeling F, Starke H: Small supernumerary <lb/>marker chromosomes – progress towards a genotype-phe-<lb/>notype correlation. Cytogenet Genome Res 2006, 112(1–2):23-24. <lb/> 22. Anderlid BM, Sahlen S, Schoumans J, Holmberg E, Ahsgren I, Mortier <lb/>G, Speleman F, Blennow E: Detailed characterization of 12 <lb/>supernumerary ring chromosomes using micro-FISH and <lb/>search for uniparental disomy. <lb/> Am J Med Genet 2001, <lb/> 99(3):223-33. <lb/> 23. Delhanty JD, Harper JC, Ao A, Handyside AH, Winston RM: Multi-<lb/>colour FISH detects frequent chromosomal mosaicism and <lb/>chaotic division in normal preimplantation embryos from <lb/>fertile patients. Hum Genet 1997, 99:755-760. <lb/> 24. Mantzouratou A, Mania A, Fragouli E, Xanthopoulou L, Tashkandi S, <lb/>Fordham K, Ranieri DM, Doshi A, Nuttall S, Harper JC, Serhal P, Del-<lb/>hanty JD: Variable aneuploidy mechanisms in embryos from <lb/>couples with poor reproductive histories undergoing preim-<lb/>plantation genetic screening. Hum Reprod 2007, 22(7):1844-53. <lb/> 25. Delhanty JDA, Handyside AH: The origin of genetic defects in <lb/>the human and their detection in the preimplantation <lb/>embryo. Human Reproduction Update 1995, 1:201-215. <lb/> 26. Wells D, Delhanty JDA: Comprehensive chromosomal analysis <lb/>of human preimplantation embryos using whole genome <lb/>amplification and single cell comparative genomic hybridisa-<lb/>tion. Mol Hum Reprod 2000, 6(11):1055-1062. <lb/> 27. Cohen O, Cans C, Mermet MA, Demongeot J, Jalbert P: Viability <lb/>Thresholds for Partial Trisomies and Monosomies – A Study <lb/>of 1,159 Viable Unbalanced Reciprocal Translocations. <lb/> Human Genetics 1994, 93(2):188-194. <lb/> 28. Reynolds A, Arora N, Donaldson A: An extremly rare, balanced <lb/>chromosome rearrangement of chromosome 22 in a normal <lb/>mother of a child with a supernumerary ring chromosome <lb/>22. J Med Gen 2004, 41(Sup 1):S58. <lb/> 29. Harper JC, Coonen E, Ramaekers FC, Delhanty JD, Handyside AH, <lb/>Winston RM, Hopman AH: Identification of the sex of human <lb/>preimplantation embryos in two hours using an improved <lb/>spreading method and fluorescent in-situ hybridization <lb/>(FISH) using directly labelled probes. Hum Reprod 1994, <lb/> 9(4):721-4. </listBibl> </back> </text> </tei>