Karyotyping of human oocytes by cenM-FISH, a new 24-colour centromere-specific technique

BACKGROUND: Metaphase II (MII) chromosome complements are difficult to karyotype. The objective of this study was to investigate the efficiency and limitations of centromere-specific multiplex fluorescence in situ hybridization (cenM-FISH), a new 24 colour FISH technique using centromere-specific probes, to analyse the whole chromosome complement within human oocytes. METHODS: Oocytes were donated by 34 patients undergoing ovarian stimulation and IVF. The MII oocytes were analysed by means of cenM-FISH, while the confirmation of results was performed by FISH and/or by analysing the corresponding first polar bodies using comparative genomic hybridization (CGH). RESULTS: A total of 30 cells, corresponding to 16 oocytes and 14 first polar bodies, were successfully karyotyped by either cenM-FISH or CGH. The incidence of aneuploidy was 25%, and eight out of nine aneuploidy events were confirmed by CGH and FISH. CONCLUSIONS: We demonstrate here for the first time that the identification of any numerical abnormality in oocytes is feasible using cenM-FISH. Despite the fact that the fixation efficiency remains low, the present results confirm the advantage of analysing the whole set of chromosomes to make an accurate estimation of the aneuploidy rate in human oocytes.     

Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization

Numerical chromosome errors are known to be common in early human embryos and probably make a significant contribution to early pregnancy loss and implantation failure in IVF patients. Over recent years fluorescent in situ hybridization (FISH) has been used to document embryonic aneuploidies. Many IVF laboratories perform preimplantation genetic diagnosis (PGD) with FISH to select embryos that are free from some aneuploidies in an attempt to improve implantation, pregnancy and live birth rates in particular categories of IVF patients. The usefulness of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. Complete karyotyping at the single cell level can now be achieved by comparative genomic hybridization (CGH). CGH enables not only enumeration of all chromosomes but gives a more complete picture of the entire length of each chromosome and has demonstrated that chromosomal breakages and partial aneuploidies exist in embryos. CGH has provided invaluable information about the extent of mosaicism and aneuploidy of all chromosomes in early human conceptuses. CGH has been applied to clinical PGD and has resulted in the birth of healthy babies from embryos whose full karyotype was determined in the preimplantation phase.     

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.     

Aneuploidy 12 in a Robertsonian (13;14) carrier: Case report

In translocation carriers, the presence of aneuploidy for the chromosomes unrelated to the rearrangement may lead to an additional risk of abnormal pregnancy or implantation failure. Consequently, it may be important to analyse not only the chromosomes involved in the rearrangement but also the rest of chromosomes. We combined spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to karyotype one unfertilized oocyte and its first polar body (1PB) from a Robertsonian translocation carrier t(13;14) aged 29 years who was undergoing IVF and preimplantation genetic diagnosis (PGD) for translocations and aneuploidy screening. Two out of four embryos were aneuploid, as a result of an adjacent segregation. The unfertilized oocyte had a normal/ balanced constitution of the chromosomes involved in the reorganization. However, this 1PB-metaphase II doublet was aneuploid for chromosome 12, the oocyte being hyperhaploid (24, X, +12) and its 1PB hypohaploid (22, X, -12). The application of CGH for the study of Robertsonian translocations of maternal origin will be useful to study imbalances of the chromosomes involved in the rearrangement, as well as alterations in the copy number of any other chromosome. The combination of PGD for translocations with aneuploidy screening could help to reduce the replacement of chromosomally abnormal embryos.     

Cellular and genetic analysis of oocytes and embryos in a human case of spontaneous oocyte activation

Unusual and consistent defects in infertility patients merit attention as these may indicate an underlying genetic abnormality, in turn necessitating tailored management strategies. We describe a case of repeated early pregnancy loss from in vivo conceptions, followed by cancelled embryo transfers after one IVF and one ICSI/PGD cycle. Following the unexpected presence of cleaved embryos at the fertilization check in the first IVF attempt, oocytes and embryos were subsequently analyzed in an ICSI/PGD case. Part of the oocyte cohort was fixed at retrieval for a cellular evaluation of microtubules, microfilaments and chromatin. The remaining oocytes were injected with sperm, and resultant embryos were biopsied for genetic analysis by fluorescence in situ hybridization (FISH), single-nucleotide polymorphism (SNP) microarray for 23 chromosome pairs, as well as with PCR for sex chromosomes. The presence of interphase microtubule networks and pronuclear structures indicated that oocytes were spontaneously activated by the time of retrieval. FISH revealed aneuploidy in all seven blastomeres analyzed, with all but two lacking Y chromosomes. Microarray SNP analysis showed an exclusively maternal origin of all blastomeres analyzed, which was further confirmed by PCR. From our multi-faceted analyses, we conclude that spontaneous activation, or parthenogenesis, was probably the pathology underlying our patient's recurrent inability to maintain a normal pregnancy. Such analyses may prove beneficial not only in diagnosing case-specific aberrations for other patients with similar or related failures, but also for furthering our general understanding of oocyte activation.     

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.     

Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies

We used fluorescent in situ hybridisation (FISH) to detect nine chromosomes (1, 13, 15, 16, 17, 18, 21, 22 and X) in 89 first Polar Bodies (1PBs), from in vitro matured oocytes discarded from IVF cycles. In 54 1PBs, we also analysed the corresponding oocyte in metaphase II (MII) to confirm the results; the other 35 1PBs were analysed alone as when preimplantation genetic diagnosis using 1PB (PGD-1PB) is performed. The frequency of aneuploid oocytes found was 47.5%; if the risk of aneuploidy for 23 chromosomes is estimated, the percentage rises to 57.2%. Missing chromosomes or chromatids found in 1PBs of 1PB/MII doublets were confirmed by MII results in 74.2%, indicating that only 25.8% of them were artefactual. Abnormalities observed in 1PBs were 55.8% whole-chromosome alterations and 44.2% chromatid anomalies. We observed a balanced predivision of chromatids for all chromosomes analysed. Differences between balanced predivision in 1PB and MII were statistically significant (P&<0.0001, chi(2) test); the 1PB was most affected. The mean abnormal segregation frequency for each chromosome was 0.89% (range 0.52-1.70%); so, each of the 23 chromosomes of an oocyte has a risk of 0.89% to be involved in aneuploidy. No significant differences were observed regarding age, type of abnormality (chromosome or chromatid alterations) or frequency of aneuploidy. Nine of the 35 patients (25.7%) whose 1PB and MII were studied presented abnormalities (extra chromosomes) that probably originated in early oogenesis. Analysis of 1PBs to select euploid oocytes could help patients of advanced age undergoing in vitro fertilization (IVF) treatment.     

Multiplex interphase FISH as a screen for common aneuploidies in spontaneous abortions

BACKGROUND: A multiplex fluorescence in-situ hybridization (FISH) strategy using chromosome-specific probes for eight chromosomes as an initial screen for chromosome abnormalities in uncultured tissues from spontaneous abortions was evaluated. METHODS: Fifty-seven prefetal spontaneous abortions were studied by karyotyping cultured cells and using FISH on uncultured cells. Two probe sets were used, identifying chromosomes 13, 15, 16, 18, 21, 22, X and Y. RESULTS: Abnormalities were detected in 53% of cases by karyotyping, and 54% of cases by FISH. FISH detected an abnormality in four of five cases where cultures failed, and in two cases where maternal cells apparently overgrew the culture. FISH missed four trisomies not identifiable with the probe sets, and one trisomy because one probe set was unscorable. FISH using these probes identified 83% of all abnormalities detected by karyotyping. CONCLUSIONS: FISH can detect abnormalities in a significant proportion of cases where the culture fails to grow or is contaminated by maternal cell growth. Multiplex FISH as an initial screen, followed by culture and karyotyping in cases where no abnormality is detected, would identify a higher proportion of chromosome abnormalities in spontaneous abortion specimens than karyotype analysis alone.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization

The use of metaphase comparative genomic hybridization (CGH) to screen all human chromosomes for aneuploidy in preimplantation embryos is hindered by the time required to perform the analysis. We report in this paper a novel approach to manufacture a DNA microarray for CGH for the detection of aneuploidy in single cells. We spotted human chromosome-specific libraries on glass slides that were depleted of repetitive sequences and tested our array CGH method in 14 experiments using either single male and/or single female lymphocytes. For the autosomes, the mean normalized ratios were all close to the expected ratio of 1.0 with overall 300/308 (97%) of the normalized ratios falling within the range 0.75 to 1.25. It was possible to deduce the correct copy number of the X chromosome in 13/14 (92.9%) separate array CGH experiments but the Y chromosome in only 4/14 (29%). We tested our microarray CGH method on a single fibroblast from each of three cell lines containing a specific chromosome aneuploidy (trisomy 13, 15 or 18) and in each case our microarray analysis was able to obtain a diagnosis based on the fact that the aneuploid chromosome gave the highest ratio (1.32, 1.27 and 1.27 respectively) with the ratios of all other chromosomes falling within the range 0.75-1.25. Requiring just 30 h, our method may be more suitable for PGD aneuploidy screening than metaphase CGH.     

Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.     

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.     

A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.      

Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis.

BACKGROUND: Robertsonian translocations carry reproductive risks that are dependent on the chromosomes involved and the sex of the carrier. We describe five couples that presented for preimplantation genetic diagnosis (PGD). METHODS: PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization (FISH) with locus-specific probes, and day 4 embryo transfer. RESULTS: Couple A (45,XX,der(14;21)(q10;q10)) had two previous pregnancies, one with translocation trisomy 21. A successful singleton pregnancy followed two cycles of PGD. Couple B (45,XX,der(13;14)(q10;q10)) had four miscarriages, two with translocation trisomy 14. One cycle of PGD resulted in triplets. Couple C (45,XX,der(13;14)(q10;q10)) had four years of infertility; two cycles were unsuccessful. Couple D (45,XY,der(13;14)(q10;q10)) presented with oligozoospermia. A singleton pregnancy followed two cycles of PGD. Couple E (45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range and low levels of aneuploid spermatozoa. PGD was therefore not recommended. No evidence for a high incidence of embryos with chaotic or mosaic chromosome complements was found. CONCLUSIONS: For fertile couples, careful risk assessment and genetic counselling should precede consideration for PGD. Where translocation couples need assisted conception for subfertility, PGD is a valuable screen for imbalance, even when the risk of viable chromosome abnormality is low.     

Mosaic supernumerary ring chromosome 19 identified by comparative genomic hybridisation.

We report the use of comparative genomic hybridisation (CGH) to define the origin of a supernumerary ring chromosome which conventional cytogenetic banding and fluorescence in situ hybridisation (FISH) methods had failed to identify. Targeted FISH using whole chromosome 19 library arm and site specific probes then confirmed the CGH results. This study shows the feasibility of using CGH for the identification of supernumerary marker chromosomes, even in fewer than 50% of cells, where no clinical or cytogenetic clues are present.     

Diagnosing and preventing inherited disease: Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization

Chromosomal aneuploidies contribute considerably to the lowpregnancy rate in in-vitro fertilization (IVF). The objectiveof this experimental work was to explore the possibility ofdetecting common aneuploidies in oocytes by polar body sampling.The study included 45 infertile patients of advanced maternalage participating in an IVF programme. The first polar bodywas removed prior to fertilization or both the first and secondpolar bodies were removed after fertilization and studied byfluorescent in-situ hybridization (FISH) using chromosome-specificprobes for chromosomes X, 18 and/or 13/21. Of 155 oocytes withFISH results, 36 demonstrated chromosomal abnormalities. Of119 oocytes predicted to be free from aneuploidy of chromosomesX, 18 and/or 13/21, 72 were normally fertilized, cleaved andtransferred in 23 treatment cycles, which resulted in two healthydeliveries and three ongoing pregnancies confirmed to be unaffectedby chorionic villous sampling. The method may appear usefulfor the detection of oocytes with common chromosomal aneuploidiesin IVF patients of advanced maternal age.     

Comparison of comparative genomic hybridization and interphase fluorescence in situ hybridization in ovarian carcinomas: possibilities and limitations of both techniques

Comparative genomic hybridization (CGH) is a valuable technique for cytogenetic analysis of solid tumors. To evaluate the reliability of CGH, we examined DNA of 10 ovarian carcinomas after CGH analysis with single- and double-locus fluorescence in situ hybridization (FISH). The FISH experiments, involving 5 chromosomes (chromosomes 3, 6, 8, 12, and 18) with different FISH probes, confirmed the CGH results in 66.2% of cases (92 of 139 investigated loci). In 4 patients, inconsistent results (41 loci) were related to polyploidy, because CGH cannot detect polyploid karyotypes. The remaining 6 discordant loci can be referred to limitations in both techniques. Re-evaluation of FISH and CGH results by one other is therefore recommended to overcome these technical artifacts. Nevertheless, CGH is of potential value in characterizing chromosomal alterations and might help in generating tumor-specific sets of FISH probes to obtain genetic information of prognostic value within a few days.     

The application of comparative genomic hybridization as an additional tool in the chromosome analysis of acute myeloid leukemia and myelodysplastic syndromes

In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) there are frequently complex karyotypes with multiple structurally altered chromosomes, many of which are marker chromosomes of unknown origin. The aim of this study was to apply comparative genomic hybridization (CGH) to cases of AML or MDS in transformation submitted for routine cytogenetic analysis to investigate whether this approach would yield any further information and, if possible, to predict which cases would benefit from CGH analysis. Nineteen cases with AML or MDS in transformation were analyzed. CGH revealed nine cases with gains or losses of chromosomal material. In six of these cases the chromosomal location of this material was not apparent from cytogenetic analysis especially when multiple markers were present. By using fluorescence in situ hybridization (FISH) with specific libraries for the chromosome regions that showed discordance between CGH and conventional cytogenetics, we were able to identify the chromosome location of material within the karyotype. In this group of six patients, four cases of an unbalanced translocation involving regions of chromosomes 5 and 17 were characterized. Three of these cases had additional abnormalities, including two cases with regions of amplification in which oncogenes are located (MYC, MLL) and one case with a dic(7;21)(p10;p10). In all six cases it was possible to characterize complex chromosomal aberrations such as derivative chromosomes, marker chromosomes, and ring chromosomes. This study demonstrates that CGH can detect true gain and loss of critical chromosome regions more accurately than conventional karyotyping in cases with very complex karyotypes, and can thus prove useful in predicting prognosis and pinpointing areas of the genome that require further study. Also, CGH can be a useful technique to identify the origin of marker chromosomes, and it can assist in choice of probes for confirmatory FISH, when there is no clue provided from the analysis of G-banded chromosomes.     

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes

BACKGROUND: Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. METHODS: After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. RESULTS: Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. CONCLUSIONS: This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.