The chromosome pattern of embryos derived from tripronuclear zygotes studied by cytogenetic analysis and fluorescence in situ hybridization*+

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1 FERTILITY AND STERILITY Copyright <!,., 995 American Society for Reproductive Medicine Vol. 6, No.6, June 995 Printed on acid-free paper in U. s. A. The chromosome pattern of embryos derived from tripronuclear zygotes studied by cytogenetic analysis and fluorescence in situ hybridization*+ Sai Ma, M.D.:j: Dagmar K. Kalousek, M.D. Basil Ho Yuen, M.B., Ch.B.:j: Young S. Moon, Ph.D.:j: University of British Columbia, Vancouver, British Columbia, Canada Objective: To detect the chromosomal complement of embryos, which developed from tripronuclear zygote, using nonradioactive centromeric probes. Design: The chromosome pattern of embryos developing from tripronuclear zygote, studied by fluorescence in situ hybridization, was compared with that of embryos studied by standard cytogenetic methods. Setting: These embryos were obtained from superovulated patients undergoing IVF treatment.. Results: We have attempted to examine the chromosomal complement of7 embryos derived from tripronuclear zygotes using both traditional cytogenetic analysis and fluorescence in situ hybridization. Ofthese 7 embryos, were analyzed with fluorescence in situ hybridization and 50 were analyzed with traditional cytogenetic analysis. For fluorescence in situ hybridization analysis, probes specific for the centromeric regions of chromosomes, 6, and were used, with results being obtained from 8 embryos. One embryo was haploid (5.6%), five were triploid (7.8%), and one was hexaploid (5.6%). Eleven (6%) embryos were mosaic. Traditional cytogenetic analysis could be performed on 5 of 50 embryos. Five (0%) were haploid, one (4%) was diploid, seven (8%) were triploid, one (4%) were tetraploid, and two were hexaploid. Nine (6%) were mosaic. Conclusion: These findings indicate that not all tripronuclear human zygotes develop into triploid embryos. This study also demonstrates the usefulness of fluorescence in situ hybridization for preimplantation diagnosis and screening for chromosome abnormalities. Fertil Steril 995;6:46-50 Key Words: Chromosome pattern, tripronuclear zygote, fluorescence in situ hybridization, standard cytogenetic analysis Cytogenetic studies of spontaneous abortions have shown that triploidy is a common finding in humans Received July 9, 994; revised and accepted January 6, 995. * Presented at the Conjoint Meeting of The American Fertility Society and the Canadian Fertility and Andrology Society, Montreal, Quebec, Canada, October to 4, 99. t Supported by grant from the British Columbia Health Care Research Foundation, Vancouver, British Columbia, Canada. :j: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology. Department of Pathology, Children's Hospital. IIReprint requests: Young S. Moon, Ph.D., Grace Hospital, Room H0, 4490 Oak Street, Vancouver, British Columbia, V6H V5, Canada (FA: ). 46 Ma et al. Chromosome analysis of human embryos (). Virtually all triploid conceptions are aborted. The prevalence oftriploidy is approximately % and 5%, in vivo and in vitro, respectively (). Although IVF of human ova offers a unique opportunity to study chromosomal abnormalities in early developmental stages, there is little information available about the karyotype of individual blastomeres derived from triploid embryos, and the data that do exist reveal complex findings (). Tripronuclear zygotes may develop into embryos with a variable chromosomal complement (i.e., triploidy, diploidy, mosaic). Traditional cytogenetic analysis of preimplantation embryos can be performed using the method developed by Tarkowski (4). However, with this ap-

2 proach it is difficult to obtain several adequate metaphases from the limited number of cells in early embryos, which interferes with mosaicism studies. Thus, there is a need for a more efficient method that allow the study of chromosomal numerical abnormalities of preimplantation embryos. The most notable recent advance in cytogenetic analysis is the development of nonisotopic fluorescence in situ hybridization techniques (5). This methodology does not require metaphase chromosomes for assessment and can be applied to interphase nuclei. Specific numerical and structural rearrangements can be detected in interphase nuclei using this molecular cytogenetic technique (6). A recent publication (7) demonstrated the feasibility of detecting specific chromosomal material in interphase nuclei of preimplantation embryos by in situ hybridization using chromosome and Y probes. In this study the chromosomal complement of preimplantation embryos derived from tripronuclear zygotes was studied. Traditional cytogenetic analysis was performed on 5 embryos and 8 embryos were studied with interphase cytogenetic analysis. The results obtained by fluorescence in situ hybridization were compared with those obtained using standard karyotyping procedures. MATERIALS AND METHODS Oocytes obtained from superovulated patients undergoing IVF treatment for infertility were collected and fertilized as described previously (8). The number of pronuclei were scored under a dissecting microscope to 0 hours after insemination. Zygotes showing more than two pronuclei were not used for ET. Seventy-two fertilized oocytes with three pronuclei were collected. The tripronuclear specimens, ranging from zygote to eight-cell stage embryos, were transferred into culture medium containing 0.4,uUmL colcemid (GIBCO, Grand Island, NY) for 6 to 4 hours to arrest cleavage at mitosis. Fifty embryos were processed for traditional cytogenetic analysis and embryos were processed for fluorescence in situ hybridization using a-satellite probes specific for, 6, and. Traditional Cytogenetic Analysis Chromosome preparation of the zygotes and embryos was performed according to the air-drying method of Tarkowski (4) with some modification. The specimens were placed in a % hypotonic sodium citrate solution for 5 to 0 minutes at 7 C. Gradual fixation involved transferring the embryos with a minimal amount of hypotonic fluid to fixative solution A (:4:5, acetic acid:methanol:distilled wa- Vol. 6, No.6, June 995 ter). The specimens were observed during fixation under a dissecting microscope and, after 0 to 40 seconds, they were placed on a precleaned slide in a minimal amount of hypotonic solution. One drop of fixative B (: methanol:glacial acetic acid) was added. The slide was then air dried and preparations were stained in a 0% Giemsa solution for minutes. Fluorescence in Situ Hybridization The zona pellucida was removed before hybridization as it prevents penetration by the probe. This involved treating the embryos with 0.5% pronase E (p-69, Sigma Chemical Co., St. Louis, MO) in culture medium for minute as observed under a microscope. After removal of the zona pellucida, the embryos were treated with hypotonic solution and fixative as described in the previous section. Standard cytogenetic preparation of lymphocyte chromosomes were used as controls for hybridization efficiency. Our hybridization protocol followed the procedure of Pinkel et al. (5) with some modification, using a satellite DNA probes for chromosomes, 6, and (Oncor Inc., Gaithersburg, MD). Briefly, slides were pretreated with 0.4,uUmL acetic anhydride before denaturation in 70% formamide-x standard saline citrate (SSC) at 70 C. Then 0,uL of hybridization mixture, containing 60% formamide, 0% 0x SSC, 50,ug of sonicated herring sperm DNA, and 5 ng/,ul of denatured DNA probe (DZ5, D6Z, DZ; Oncor) were placed on the slide, coverslipped, and sealed. Hybridization of probe to the genomic DNA proceeded overnight at 7 C. After hybridization, slides were washed for 0 minutes in 50% formamide- SSC at 8 C. The probe was detected by sequential incubations in 5,ug/mL avidin-fluorescein isothiocyanate conjugate (FITC), 5,ug/mL biotinylated antiavidin D, and 5,ug/mL avidin-fitc. The slides were washed in a phosphate detergent buffer, counter stained with,ug/ml propidium iodide in antifade solution (0.% p-phenyldiamine dihydrochloride), and coverslipped. Nuclei from each embryo were examined with a Nikon fluorescence microscope with filter module BA at 600x magnification (Nikon Inc., Garden City, NY). The hybridized FITClabeled probe was visualized as a bright yellow hybridization signal. In this study, we have used the number of hybridization signals for scoring ploidy. The DNA of each human chromosome occupies a discrete focal territory within an interphase nucleus. Therefore, each hybridization signal indicates the presence of a specific centromeric DNA. Haploidy, diploidy, triploidy, and tetraploidy would be represented by,,, and 4 hybridization signals, respectively. Ma et al. Chromosome analysis of human embryos 47

3 Table Results of Cytogenetic Analysis of 5 Embryos* Derived From Human Tripronuclear Zygotes Total Stage of no. of Karyotype Case embryos Cytogenetic results embryost Haploidy Two-cell 0 (one cell) (one cell) (one cell) 4 (one cell) 5 Eight-cell (one cell) 5 (0) Diploidy 6 46, (one cell) 7 5/7 (three cells) 8 4/40 (two cells) 9 Eight-cell 4/44 (two cells) 0 Eight-cell 44/45 (two cells) Eight-cell 6/50 (three cells) 6 (4) Triploidy Zygote 69,Y (one cell) 69, (one cell) 4 69, (one cell) 5 69, (one cell) 6 69, (one cell) 7 8 (one cell) 8 Zygote 78 (one cell) 9 9/40/69 (three cells) 0 67 (two cells) 9 (6) Tetraploidy Zygote 9 (one cell) 8/9/90 (three cells) Two-cell /90 (two cells) () Hexaploidy 4 Zygote 8 (one cell) 5 Zygote 8 (one cell) (8) * When sex chromosome is not indicated, the preparation did not allow identification of individual chromosome. t Values in parentheses are percentages. RESULTS Traditional Cytogenetic Analysis Fifty embryos were processed for traditional cytogenetic analysis. Results were available from 5 zygotes or embryos, as summarized in Table. Nine embryos (6%) were of triploid range, including five triploidy, two hypertriploidy, two mosaic, whereas six (4%) were within diploid range, with five of them being mosaic. Three (%) specimens showed tetraploidy, of which two were mosaic. Two zygotes (8%) with a modal chromosome number of 8 were classified as hexaploid. Five embryos (0%) showed haploid complements, including three haploidy, one hypohaploidy, and one hyperhaploidy. It is noteworthy that all embryos with more than one analyzed cell showed mosaicism. Fluorescence In Situ Hybridization Analysis Interphase nuclei of human preimplantation embryos were studied by fluorescence in situ hybridization using, 6, and a-satellite DNA probes. Control lymphocyte chromosome preparations showed 95% to 00% efficiency for all three probes used. The three probes used in this study did not show cross-hybridization either in sample or control group. 48 Ma et al. Chromosome analysis of human embryos Results were obtained from 8 embryos (Table ). Cases, 8, and 4 were evaluated using an chromosome-specific probe. Case 4 was analyzed using a chromosome I-specific probe, whereas cases 6 to, 5 to 7, and 9 to 4 were evaluated using a centromeric probe specific for chromosome 6. The results from two cases were not included for interpretation because hybridization with the chromosome probe showed either one or two signals. In these embryos, nuclei showing less than three signals may contain a Y chromosome. Interpretation of the number of hybridization signals indicated that case 6 was haploid. Cases 7, 8, 9, 0,, and were within diploid range with all of them being mosaic. Cases, 4, 5, 6, and 7 were triploid, whereas cases 8, 9, 40, 4, and 4 were triploid mosaic (Fig. la). Case 4 was hexaploid (Fig. IB). There were no signals recorded in only eight blastomeres. These negative results may be caused by hybridization failure rather than absence of chromosomes in the nuclei. DISCUSSION Traditional cytogenetic techniques allow the identification of individual chromosomes and accurate diagnosis of various chromosomal aberrations in preimplantation embryos. These studies, however, currently are limited by the difficulty to obtain analyzable metaphases. Fluorescence in situ hybridization using a-satellite centromeric probes produces distinct signals that can be detected easily and counted in interphase nuclei. In our study, cytogenetic information could be obtained from only 5 of 50 tripronuclear embryos, whereas 8 of embryos could be evaluated by fluorescence in situ hybridization. These results indicate that the application of in situ hybridization is useful in the study of chromosomal abnormalities in preimplantation embryos. Triploidy results from a number of different mechanisms including dispermy, fusion of a diploid sperm with a haploid oocyte, or fertilization of a diploid oocyte by a haploid sperm. Most probably, triploidy arising from one haploid and one diploid maternal or paternal nucleus will never be recognized in routine IVF procedures in which triploidy is based only on morphological observation of three pronuclei. Whether a failure in the second maternal meiosis can lead to an extra maternal pronuclei is unknown; if not, the observation of three pronuclei implies the penetration of two spermatozoa (). The subsequent karyotype oftripronuclear zygote has been described in detail by Kola et al. (9) and observed in other studies. Kola et al. (9) found 4% embryos had triploid pattern, whereas Pieters et al. () stated that only 4% of embryos were completely triploid.

4 Table Results of Fluorescence In Situ Hybridization Analysis of 8 Embryos Derived From Human Tripronuclear Zygotes No. of hybridization nuclei signals per nuclei scored Interpretation Case Stage of embryos Probe 0 Haploidy Diploidy or mosaic Eight-cell Eight-cell Zygote Triploidy or mosaic Hexaploidy Figure Interphase cytogenetic analysis. (A), One blastomere containing three signals, the other two showing one signal in each blastomere from a six-cell embryo with a-satellite 6 probe. (B), Anuncleaved embryo with six signals of a-satellite probe. Vol. 6, No.6, June Zygotes showing three pronuclei are thought to be karyotypically triploid. In our study, using both traditional and interphase cytogenetic analysis, only 7.9% (/4) analyzed embryos were found to be triploid. Cases 4 and 5 showed a S-chromosome complement and case 4 presented six signals in the fluorescence in situ hybridization study. In these cases, it is assumed that a triploid zygote underwent a chromosome endoreduplication or no spindle formation after DNA synthesis. Tripronuclear embryos also can develop into embryos with a normal diploid chromosome complement. One four-cell stage embryo (case 6) had a normal 46, karyotype. Development of diploid embryos from tripronuclear zygotes requires extrusion of a haploid nucleus (9) followed by a normal division involving 46 chromosomes with a normal bipolar spindle. Kola et al. (9) found that 4 of 9 embryos cleaved from tripronuclear human zygotes had 46, karyotypes. Haploidy was detected in both cytogenetically and fluorescence in situ hybridization-analyzed embryos (cases to 5 and 6). Haploidy in a tripronuclear zygote may be caused by exclusion of two haploid sets entirely from the metaphase plate at first cleavage or their degeneration. This condition could be similar to parthenogenesis. It also is possible that two cytoplasmic vacuoles are misinterpreted as pronuclei. In this situation, a modal chromosome number of would be the result of parthenogenesis. Whereas the use of human embryos for experimental purposes continues to be restricted because of ethical reasons, embryos resulting from multipronuclear zygotes are useful for the study of early prema et at Chromosome analysis of human embryos 49

5 implantation stages. The above results support the conclusion that the chromosomal complement established by analysis ofindividual blastomere of triploid human embryos is unreliable because mosaicism is common (). In the studies reported in the literature, most of the data were based on single-cell analysis (, 9, 0) and therefore it should be kept in mind that these embryos' single cells do not reflect the constitution of the whole embryo. In this study, many different types of mosaicism developing from tripronuclear zygotes were identified. These included 4.7% (/4) of mosaic tetraploidy, 6.% (7/4) of mosaic triploidy, and 5.6% (/4) of mosaic diploidy. Pieters et al. () also found mosaicism to be common in the embryos originating from tripronuclear zygotes. The mosaicism most likely arises from abnormal spindle formation, which leads to disorganized movement of chromosomes to the poles (9). Accurate distinction between mosaic and nonmosaic embryos requires the analyses of two or more metaphases by cytogenetic or interphase nuclei analysis. Among the 5 embryos analyzed cytogenetically, 64% showed only one analyzable mitosis (Table ), 0% had two analyzable mitoses, and 6% had three. All embryos with two or more metaphases showed mosaicism. This suggests that the frequency of mosaicism is underestimated in this study, because most embryos produced only one metaphase. Analysis of many nuclei by fluorescence in situ hybridization also is indicative of a high frequency of mosaicism in tripronuclear embryos. Previous studies of human blastomeres have shown that fluorescence in situ hybridization may become the technique of choice for preimplantation diagnosis of -linked disease and chromosomal abnormalities (7,, ). However, it is difficult using only one probe to differentiate between true mosaicism or artifact associated with poor hybridization. In the present study, cases 7 to and 8 to 4 showed less than three hybridization signals in some nuclei. There are two possible interpretations for these results: first, they may be truly mosaic as seen in cytogenetic studies or, second, this could be explained by weak hybridization resulting in signals too faint to detect. The use of multiple probes simultaneously for fluorescence in situ hybridization analysis of the preimplantation embryos will provide an efficient tool for detection of the mosaicism. In conclusion, this study provides further evidence that most embryos derived from tripronuclear zygotes have variable chromosome patterns and that only approximately 7.9% of them are triploid. The frequency of triploidy detected in embryos by fluo- rescence in situ hybridization is similar to cytogenetic studies. Analysis of two or more nuclei per metaphase from each embryo reveals a high incidence of mosaicism in embryos developing from tripronuclear zygote. This study also demonstrates the main advantage of the use of fluorescence in situ hybridization for chromosomal evaluation of preimplantation embryos: the ability to analyze several individual interphase nuclei from very early embryos. However, a combination of two or more probes is necessary to increase the reliability and the accuracy of fluorescence in situ hybridization. Acknowledgments. We thank Brenda Lomax, B.Sc., and Brian Kuchinka, M.Sc., for their technical assistance; Pathma Rajamahendran, B.Sc., Ms. Cindy Trousil, and Ms. Tammy Chutskofffor their willing co-operation with the collection of the material; and Karen Harrison, Ph.D., and Irene Barrett, B.Sc., for their assistance in the preparation of this manuscript. REFERENCES. Lauritsen JG. The cytogenetics of spontaneous abortion. Res Reprod 98;4:-4.. Angell RR. Chromosome abnormalities in human preimplantation embryos. Prog Clin BioI Res 989;94:8-7.. 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