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REGULAR ARTICLE Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines Yury Verlinsky a,1, Nicolas H. Zech a,b,⁎,1, Nikolai Strelchenko a, Valeri Kukharenko a, Artem Shkumatov a, Zev Zlatopolsky a, Anver Kuliev a

a Reproductive Institute, 2825 North Halsted, , IL 60657, USA b Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland

Received 19 May 2008; received in revised form 9 July 2008; accepted 14 July 2008

Abstract There are several sources from which human (hESC) lines can be generated: surplus after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during of embryos carrying such errors. © 2008 Elsevier B.V. All rights reserved.

than to transfer them back to the oocyte donor (Liao, 2005). Introduction Still, the ethical aspects of using even surplus embryos to generate hESC lines are discussed controversially (Boer, Human embryonic stem cells (hESC) are usually produced 1999; Meyer, 2000; Bahadur, 2003; Ohara, 2003; Reichhardt through the use of surplus embryos derived from in vitro et al., 2004; Devolder, 2005; Edwards, 2005; Pennings, 2006; fertilization procedures. In many countries, legislation European Group on Ethics in Science and New Technologies, influences the availability of embryos, with some countries Opinion No. 15, Ethical aspects of human stem cell research even banning the creation of embryos for any other purpose and use: http://ec.europa.eu/european_group_ethics/ docs/avis15_en.pdf, accessed on 14 November 2000). ⁎ Corresponding author. For example, laboratories in the using hESC E-mail address: [email protected] (N.H. Zech). lines derived from surplus embryos after 9 August 2001 are 1 These authors contributed equally to this work. denied federal funds for research. At the same time, state

1873-5061/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scr.2008.07.002 PGD for chromosomal aneuploidies and establishment of hESC lines 79 and private agencies remain free to fund the acquisition of Results embryos for hESC research, including the derivation of new hESC lines. The supply of available embryos remains short of We defined and studied four groups of aneuploidies: demand, even in those countries where hESC research is Group I consisted of exclusively PB diagnosis and permitted. In the United States, only around 2.8% of surplus blastocysts originating from zygotes that tested positive for embryos are available for research (Hoffman et al., 2003), 2% monosomy, trisomy, or multiple errors. Such chromosomal in Canada (Baylis et al., 2003), and only 0.4% in Switzerland abnormalities comparably originate from meioses I and II (Koeferl-Puorger et al., 2006), because of the restrictive (Kuliev and Verlinsky, 2004). Usually, all cells in the resulting legal regulations. embryos are abnormal for the same chromosome(s). In Group There are several other sources from which hESC lines can I, 87 blastocysts from a total of 174 karyotypically abnormal be generated, for example, the use of one- and three- blastocysts were trisomic, 45 were monosomic, and 42 pronuclear zygotes, early arrested or highly fragmented showed multiple abnormalities. Four blastocysts in this embryos that have reached the blastocyst stage, or other- group had an ambiguous result (trisomy 13, trisomy 21, wise abnormal embryos with gene and chromosome disorders monosomy 21, and monosomy 22). obtained through preimplantation genetic diagnosis (PGD) Group II consisted of exclusively PB diagnosis and (Verlinsky et al., 2005, 2006; Lerou et al., 2008). Human ESC blastocysts originating from zygotes showing a balanced lines produced from aneuploid embryos provide an extre- karyotype. It is known that approximately 43% of oocytes mely valuable source for research on the primary effects of with meiosis I errors also have sequential meiosis II errors, chromosomal abnormalities (Verlinsky et al., 2008). resulting in 33% of cases in karyotypically balanced zygotes. We report on the efficiency of establishing hESC lines from The cause for the formation of such balanced zygotes is not blastocysts with euploid and aneuploid karyotypes after yet understood. We suggest that the underlying mechanism is sequential testing of both polar bodies (PBs) and blastomere an “aneuploidy rescue” event in female meiosis. This event analysis on day 3. Our findings suggest that it seems reason- may be similar to the well-known phenomenon of “trisomy able to perform sequential polar body and blastomere testing rescue” in postzygotic development, which may on embryos prior to the initiation of hESC culture for the result in uniparental disomy and imprinting disorders (Kuliev derivation of new lines. We argue that embryos with euploid and Verlinsky, 2004). Nonetheless, such embryos either have and aneuploid karyotypes may show significantly different a stable diploid karyotype or may show a predisposition to success rates for establishing hESC lines owing to the impact of further postzygotic and, as a result, mitotic nondisjunction meiotic and mitotic nondisjunction events (Verlinsky et al., events. Group II contained 37 blastocysts, all carrying a 2008). It may, therefore, generally prove very difficult to balanced chromosomal error. obtain abnormal hESC lines for scientific study from aneuploid Group III consisted of PB1 and/or PB2 karyotypically PGD embryos, which will limit our ability to study the bio- normal and blastomere biopsy showing chromosomal errors. logical consequences of chromosomal abnormalities. Such chromosomal abnormalities may arise from mitotic Furthermore, the success rates for generating aneuploid errors in one or more blastomeres of an otherwise karyoty- cell lines originating from fertilized oocytes carrying a pically normal embryo. These embryos can be classified as prezygotic nondisjunction error seem to mirror the mis- being mosaic, with a mix of karyotypically normal and carriage rates seen for resulting from embryos abnormal cells. Group III incorporated 99 karyotypically carrying such errors. abnormal blastocysts, of which 35 were diagnosed as

Table 1 Comparison of success rates for generating hESC lines between the four different groups Group I Group II Group III Group IV No. of karyotypically abnormal blastocysts 174 37 99 139 No. of embryos with ambiguous results 4 ––– No. of hESC lines 7 8 9 17 No. of euploid hESC lines from ambiguous results 4 ––– No. of euploid hESC lines from proven errors 0 8 8 15 No. of hESC lines from proven monosomies and trisomies 3

Success rate per blastocyst No. of hESC lines (%) 7/174 (4.0) ⁎ 8/37 (21.6) 9/99 (9.1) 17/139 (12.2) No. of hESC lines (%) from proven monosomies and trisomies (PB) 3/128 (2.4) ⁎⁎ ––– No. of hESC lines (%) from proven errors 2/170 (1.2) ⁎⁎⁎ ––– % euploid hESC lines from proven errors 0% 100% 89% 88% Group II vs Group III, P=0.09. Group II vs Group IV, P=0.22. Group III vs Group IV, P=0.54. ⁎ Group I vs Group II, Pb0.01; vs Group III, P=0.12; vs Group IV, Pb0.05. ⁎⁎ Group I vs Group II, Pb0.01; vs Group III, Pb0.05; vs Group III, Pb0.01. ⁎⁎⁎ Group I vs Group II, Pb0.01; vs Group III, Pb0.01; vs Group III, Pb0.01. 80 Y. Verlinsky et al. trisomic, 47 as monosomic, and 17 as having multiple chro- karyotyping and the reported prevalence in live births mosomal abnormalities. (Snijders et al., 1994; Snijders et al., 1995; Snijders et al., Group IV consisted of embryos that tested positive for 1999; Halliday et al., 1995). All these studies found that the chromosome errors after blastomere diagnosis alone. We prevalence of trisomy 21 is influenced by maternal age and is assume that the major sources of chromosomal abnormalities higher in early pregnancy than in live births. Furthermore, in embryos are meiosis I and II errors, with mitotic errors the estimated rates of fetal loss show an overall decrease playing a tangential role (Kuliev and Verlinsky,2004). However, over the course of pregnancy, with the highest loss rates as data on PBs are missing in Group IV,we can only speculate on estimated to occur before 10 weeks. the underlying cause for the chromosomal abnormalities: Similar methods were used to produce risk estimates for meiotic error with predisposition to further mitotic errors, other chromosomal abnormalities (Snijders et al., 1995). For aneuploidy rescue of an abnormal zygote during the first example, the rate of intrauterine lethality for trisomies 18 and mitotic divisions, or primarily postzygotic mitotic errors of 13 between 12 and 40 weeks was calculated to be about 80%. single cells. In Group IV, 44 of 139 karyotypically abnormal The vast majority (95%) of all observed cases of Down blastocysts were diagnosed as trisomic, 55 as monosomic, and syndrome result from meiotic nondisjunction events (88% 40 as carrying multiple errors. originating from nondisjunction in the maternal gametes and As summarized in Table 1, the overall success rate for 8% from nondisjunction in the paternal gametes), 2–3% from generating karyotypically normal and abnormal stem cell Robertsonian translocation, and 1–2% from mosaicism lines from blastocysts originating from chromosomal abnor- (either postzygotic nondisjunction during early embryo cell mal zygotes and embryos differed statistically significantly division, leading to a fraction of the cells with trisomy 21, or between Group I (4.0%), Group II (21.6%), and Group IV through postzygotic aneuploidy rescue, by which some of the (12.2%). Excluding those blastocysts in Group I originating cells of the embryo revert to the normal chromosomal set). from zygotes with ambiguous results, the success rate for is rarely due to a duplication of a portion of generating hESC lines including only monosomies and chromosome 21 (Down syndrome occurrence rates (NIH), trisomies dropped to 2.4% with 0% chromosomally normal retrieved on 12 April 2008: http://www.nichd.nih.gov/ lines and differed significantly from the other three groups. publications/pubs/downsyndrome.cfm#TheOccurrence). One hundred percent of hESC lines derived from 37 Considering the available estimates of pregnancy loss due chromosomally abnormal blastocysts in Group II had a normal to aneuploidies as outlined above, the true rate of pregnancy karyotype at least up to passages 10–15. In Groups III and IV, loss with aneuploid embryos resulting from prezygotic where PB diagnosis showed a normal result and blastomere nondisjunctions if extrapolated from our findings could be testing was abnormal (Group III) or where only blastomere as high as 97.6% for both trisomy 21 and trisomy 13, with only biopsy was performed (Group IV), all but three resulting stem 2.4% reaching live birth. Although postzygotic aneuploidy cell lines were karyotypically normal (89 and 88% overall rescue may occur in embryos aneuploid from the onset, we success rate in Groups III and IV, respectively, for generating observed no karyotypically normal cells in all three aneu- karyotypically normal hESC lines). Two abnormal hESC lines ploid hESC lines (trisomy 13, trisomy 21, and monosomy 22). had the same sex chromosome abnormality as the tested Further follow-up in these abnormal hESC lines may provide embryo, and one presented a different anomaly compared to more information on the true incidence of pre- and the original PGD analysis (47XY+21 in blastomere analysis, postzygotic aneuploidy rescue. 46XY/49XXY+12+15 in the resulting hESC line). Group IV may represent a mix of embryos, originating from prezygotic meiotic and postzygotic mitotic errors, with the overall success rate for establishing hESC lines similar to that Discussion of Group III (9.1%) (P=0.54) and in between Groups I and II. As all but one of the hESC lines from Group III and all but two from At least 50% of oocytes have a meiotic I or II error (Verlinsky et Group IV were karyotypically normal (89 and 88% normal al., 2001; Kuliev et al., 2005). In this study, the success rate of stem cell lines for Groups III and IV, respectively), either a establishing hESC lines from aneuploid zygotes, including postzygotic mitotic aneuploidy rescue occurred or otherwise those with trisomies and monosomies, was as low as 2.4%. Our normal cells had a survival advantage over chromosomally results clearly show that in the process of generating hESC unstable cells in the long run (Peura et al., 2008). Our results lines it would be useful to perform testing of both PBs and one show that only a small fraction of embryos with a proven blastomere from day 3 embryos to predict the success rate of aneuploidy after PB testing can generate aneuploid hESC lines. establishing hESC lines from embryos that harbor prezygotic From this it can be deduced that survival chances are very low meiotic errors (Kuliev et al., 2005). for karyotypically abnormal cells in the mosaic embryos in As of now, only crude estimates on pregnancy loss due to Groups III and IV, irrespective of the underlying cause for the such aneuploidies are available. There are, for example, mosaicism: meiotic error with predisposition to further estimates based on surveys that calculate maternal age- mitotic errors, aneuploidy rescue of an abnormal zygote specific incidences of trisomy 21 at birth (Hecht and Hook, during the first mitotic divisions, or primarily postzygotic 1994). In other cases, estimates are based on comparing the mitotic errors of single cells. In most cases, only those cells birth prevalence of trisomy 21 (Hecht and Hook, 1994) to its with a normal chromosomal makeup probably contributed to prevalence in women undergoing second-trimester amnio- the newly created stem cell lines. However,regarding the two centesis or first-trimester chorionic villus sampling. Rates of karyotypically abnormal hESC lines observed in Group IV, it spontaneous fetal death between different gestations and could well be that selection of the fastest growing colonies delivery at 40 weeks were estimated on the basis of both the during the derivation phase of the lines may have resulted in observed prevalence in pregnancies that had antenatal fetal adapted chromosomally abnormal hESC lines. PGD for chromosomal aneuploidies and establishment of hESC lines 81

It is well accepted that the efficiency of hESC production a large signal or overlapping signals, loss of nuclear material is influenced by a number of factors, in particular, the quality during fixation, or poor hybridization. of the blastocysts and the degree of lethality of each type of aneuploidy at different stages of preimplantation develop- Categories of aneuploid embryos ment. Furthermore, we are aware of the fact that the original diagnosis of biopsied PBs and/or blastomeres was Four groups of aneuploidies were studied: Group I, exclu- performed by FISH and was limited to the analysis of 10 of the sively PB diagnosis and blastocysts originating from zygotes 24 chromosomes. It might, therefore, be argued that any that tested positive for monosomy, trisomy, or multiple undiagnosed aneuploidies in the remaining 14 chromosomes errors; Group II, exclusively PB diagnosis and blastocysts could have influenced hESC production, particularly if their originating from zygotes showing a balanced karyotype; incidence is disproportionate between the four groups of Group III, PB1 and/or PB2 karyotypically normal and blastocysts. blastomere biopsy showing chromosomal errors; and Group As shown earlier by our group (Kuliev et al., 2005), the IV, embryos tested positive for chromosome errors after errors of the tested chromosomes represent the majority of blastomere diagnosis alone. chromosomal abnormalities in the embryo, as remaining untested chromosomes are very rarely involved in meiotic Isolation and characterization of hESC lines errors. Additionally, a large proportion of cases represents complex abnormalities, with an extremely high frequency Depending on the developmental stage of these aneuploid of additional errors in meiosis I and meiosis II, as well as embryos, different techniques for the establishment of hESC sequential meiosis I and meiosis II errors of the same and lines were used, as described previously (Strelchenko et al., additional chromosomes involved. Furthermore, the previous 2004). The initial disaggregation of the cells (passage 0) was data on comparative genomic hybridization (CGH) showed no performed approximately 8–14 days after growth on the significant difference between FISH and CGH data, although feeder layer, by treating the cells with EDTA and cutting and the CGH technique has still not been improved to the extent transferring the soft cell clumps into a new dish with the that it is sufficiently reliable in single cells and as such cannot feeder layer. Fast-proliferating colonies with hESC-like be used for practical purposes. morphology were isolated and propagated further. Within As for the blastocyst quality, our preliminary data show no the next 2–5 passages, the uniformly proliferating cells were significant differences between blastocysts with normal and selected, and colonies of established ESC lines were those with abnormal karyotypes, neither in this study nor in passaged using EDTA. This was followed by the harvesting our previous publication on this subject (Verlinsky and Kuliev, procedure with a cell lifter, as described previously 2005). (Strelchenko et al., 2004). The data presented suggest the prognostic value of The cell lines were tested for the following hESC markers: sequential PB1 and PB2 analysis in addition to blastomere alkaline phosphatase, stage-specific antigens SSEA-3 and SSEA- biopsy on day 3, not only for the preselection of chromoso- 4, high-molecular-weight glycoproteins or tumor rejection mally normal embryos suitable for transfer, but also for the antigens, TRA-1–60 and TRA-1–80, and Oct-4. Detection was highest success rate in establishing hESC lines. Furthermore, achieved using both polyclonal antibodies and the Gene Choice abnormal hESC lines from aneuploid PGD embryos might One Tube RT-PCR kit (Vector Laboratories, Burlingame, CA, prove very difficult to produce, thus limiting our ability to USA), as described previously (Strelchenko et al., 2004). study the biological consequences of chromosomal Regular chromosomal analysis was performed using the G- abnormalities. banding technique on 10–20 cells of the established hESC lines before freezing at passage 10–15. Methods Statistical analysis Preimplantation genetic diagnosis A double-sided Fisher's exact test was conducted to compare The preimplantation embryos for the establishment of hESC the different groups, using SPSS for Windows 11.0 (Chicago, lines with genetic disorders were obtained from PGD cycles IL, USA). Percentages were rounded up to the first decimal that had been performed either by sequential PB1 and PB2 place. P values below 0.05 were considered to be statistically biopsy followed by the removal of one blastomere from day 3 significant. embryos or by single blastomere biopsy alone on day 3, as described elsewhere (Verlinsky and Kuliev, 2000). Following References both FISH analysis of the biopsied materials using probes specific for chromosomes 13, 16, 18, 21, and 22, and rehybridization using specific probes for 9, 15, 17, X, and Y Bahadur, G., 2003. The moral status of the embryo: the human chromosomes in blastomere analysis, the unaffected embryo in the UK Human Fertilisation and (Research Purposes) Regulation 2001 debate. Reprod. Biomed. Online 7, embryos were transferred back to patients. The chromoso- 12–16. mally abnormal ones were used for derivation of hESC lines, Baylis, F., Beagan, B., Johnston, J., Ram, N., 2003. Cryopreserved according to informed consent as approved by the IRB of the human embryos in Canada and their availability for research. Reproductive Genetics Institute. 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