Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production

Transcription

1 SPECIAL CONTRIBUTIONS Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production Santiago Munné, Ph.D., a Esther Velilla, M.Sc., a Pere Colls, Ph.D., a Mercedez Garcia Bermudez, Ph.D., a Mohan C. Vemuri, Ph.D., a Nury Steuerwald, Ph.D., b John Garrisi, Ph.D., c and Jacques Cohen, Ph.D. a a Reprogenetics, LLC, West Orange, New Jersey; b A.R.T. Institute of New York and New Jersey, West Orange, New Jersey; and c Saint Barnabas Medical Center, Livingston, New Jersey Objective: To ascertain whether embryos classified by preimplantation genetic diagnosis (PGD) for infertility as abnormal and then plated to obtain stem cells would self-correct partially or totally in culture, producing disomic stem cells. Design: Prospective study to determine the chromosome status of embryos on day 3 and 6, as well as cultured cells derived from inner cell masses from the same embryos when cultured up to day 12. Setting: Research laboratory. Patient(s): Patients undergoing PGD of aneuploidy. Intervention(s): Of 142 embryos classified by PGD for aneuploidy as abnormal, 50 were cultured to the blastocyst stage. At that stage a fraction of the embryos underwent trophectoderm biopsy to reconfirm the PGD diagnosis. After further co-culture with feeders up to day 12, 34 embryos attached to the feeder cells. Of those, 24 were analyzed by fluorescence in situ hybridization (FISH) and the rest for the expression of Oct-4, SSEA-3, SSEA-4, TRA1-60, and TRA1-80. Main Outcome Measure(s): Disomic cells obtained from trisomic embryos. Result(s): Analysis by FISH of day-12 cultures showed that 7 were totally normal, 6 were mostly abnormal, and 11 had experienced some chromosome normalization, having between 21% and 88% normal cells. Day-12 culture was positive for Oct-4 expression by reverse transcriptase polymerase chain reaction analysis and for SSEA-3, SSEA-4, TRA1-60, and TRA1-80 by immunocytochemistry. Conclusion(s): Chromosome self-normalization occurs in a significant proportion of chromosomally abnormal embryos, possibly because of the loss of a chromosome in trisomic cells after blastocyst stage. Thus chromosomally abnormal embryos are a potential source of disomic stem cells. Not all chromosomally abnormal embryos self-corrected. Abnormal stem cells that might be derived could be used as models to study the effect of chromosomal abnormalities on human development. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Trisomy, preimplantation genetic diagnosis, mosaicism Human embryonic stem cells (hescs), which are derived from the inner cell mass of the blastocyst, are pluripotent, have a normal karyotype, and can be maintained indefinitely in culture (1, 2). These cells might be used for drug discovery, the study of early human development, and as an unlimited source of cells for transplantation therapy. Thomson and his group (3) described for the first time the isolation of hesc lines. This was confirmed later by Reubinoff et al. (4), who reported that hescs could differentiate in vitro into different somatic lineages. Received February 7, 2005; revised and accepted June 30, Reprint requests: Santiago Munné, Ph.D., Reprogenetics, LLC, 101 Old Short Hills Road, Suite 501, West Orange, New Jersey (FAX: ; munne@embryos.net). The use of normal embryos or the creation of human embryos for stem cell research has been controversial, and a ban on research with government funds has been applied in the United States, yet some individual states, such as California and New Jersey, have condoned this type of experimentation. In some countries, such as the United Kingdom, hesc research has been regulated and is acceptable. For this reason, we hypothesized that trisomic embryos could be a source of chromosomally normal cells if the tissues become mosaic in culture. For instance, uniparental disomy has been extensively reported in prenatal diagnosis work and its origin explained as the loss through mosaicism of one chromosome in a trisomic cell (5 7). In trisomic embryos, the loss of the extra chromosome is known as trisomic zygote rescue (8) Fertility and Sterility Vol. 84, No. 5, November /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 The objective of this study was to prove that diploid cells could be obtained from the extended culture of trisomic embryos cultured in monolayer. MATERIALS AND METHODS Embryos classified by preimplantation genetic diagnosis (PGD) as chromosomally abnormal were used for this study. Institutional review board approval for this study was obtained, and written consent was provided by each patient. Determination of Chromosome Normalization On day 3 of development, each embryo had a single cell biopsied (9) unless the nucleus could not be found after fixation, when a second cell was biopsied. Fixation was performed as described previously (10). For both the PGD analysis and the reanalysis, cells were analyzed for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22 according to previously published fluorescence in situ hybridization (FISH) protocols (11, 12). After analysis, if the specific signals for a chromosome were not clearly diagnosable because they were too close to each other, a third hybridization with a telomeric probe for that chromosome was used, as described by Colls et al. (13). This approach was used to determine whether the close signals represented one or two chromosomes. On the basis of these results, embryos classified as normal by PGD were replaced, whereas certain embryos classified as chromosomally abnormal were isolated. Specifically, trisomic and monosomic embryos were obtained for this study. Embryos classified by PGD as chromosomally abnormal were cultured in sequential media until day 6. At that time, some blastocysts had their trophectoderm biopsied with laser ablation, according to previously described protocols (14). This step was performed to confirm that the embryo was chromosomally abnormal. The biopsied trophectoderm cells were fixed and analyzed by FISH, following the same methods as used for PGD. In those embryos that were laser biopsied, the remainder of the embryo was plated onto mouse embryonic fibroblast cells (American Type Culture Collection-STO cell line) previously mitotically inactivated by mitomycin C on gelatincoated tissue culture dishes. Maintenance of mouse feeder cells was performed according to cell supplier s recommendations. The culture medium consisted of Dulbecco s modified Eagle medium (without sodium pyruvate, glucose 4,500 mg/l; Life Technologies, Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (Life Technologies), 0.1 mmol/l -mercaptoethanol, 1% nonessential amino acids, 2 mmol/l L-glutamine, 50 UI/mL penicillin, and streptomycin 50 UI/mL. At the time of embryo culture the medium was supplemented with human recombinant leukemia inhibitory factor (Sigma Chemical, St. Louis, MO) at 2,000 UI/mL and basic fibroblast growth factor 4 ng/ml. Embryos were cultured in this system until day 12, and then the human cells were fixed and analyzed by FISH, following the method described above. Some embryos were not analyzed by FISH on day 6 and were cultured to day 12 as described above. The reason was the potential invasiveness of the procedure. In this initial study, trophectoderm and inner cell mass were not independently fixed for further FISH analysis. For those embryos with results on all three sampling days, the different FISH results obtained on day 3, 6, and 12 were compared to determine whether the PGD diagnosis was correct (day 3 vs. day 6), and to determine whether there was cell correction through extended culture (day 6 vs. day 12). In the past, we have reported FISH error rates of approximately 7% 10% (12) when the PGD analysis was based on a single cell. In some of the studied embryos here, we did not reconfirm on day 6 for technical reasons. Misdiagnosis by PGD of an abnormal cleaved embryo can be excluded when there are still many abnormal cells in the day-12 culture. We have set this proportion arbitrarily at 20%, a rate that is at least double that of the theoretical FISH error rate. Determination of Stem Cell Presence Octamer-binding transcription factor 4 (Oct-4) is a POU domain transcription factor expressed in undifferentiated hescs and downregulated upon differentiation (15). For those cell cultures reaching day 12, stem cell Oct-4 marker was analyzed. To monitor the expression of Oct-4, reverse transcriptase polymerase chain reaction (RT-PCR) was carried out on colonies derived from abnormal embryos. Total RNA was isolated with an RNaqueous kit (Ambion, Austin, TX) according to the manufacturer s instructions. First-strand complementary DNA was synthesized by priming with random hexamers, as previously described (16). Briefly, reverse transcription was performed by the addition of 12 L containing 4 L 25 mmol/l MgCl 2,2 L 10 PCR buffer (Perkin Elmer, Foster City, CA), 4 L deoxyribonucleoside triphosphates (dntps) 2 mmol/l, 1 L ribonuclease inhibitor (20 IU/ L), and 0.5 L Mouse Moloney murine leukemia virus reverse transcriptase, and incubated at 37 C for 60 minutes. Oct-4 primers sequences were obtained from Xu et al. (15). One L of complementary DNA was used directly for PCR per 25 L PCR reaction mixture, comprising 10 PCR buffer, 1.5 mmol/l MgCl 2, 200 mol/l dntps, 20 mol/l primers, and 1.25 U of Taq polymerase. Reactions were subjected to 40 PCR cycles of each: denaturation at 94 C for 30 seconds, annealing at 55 C for 30 seconds, and strand elongation at 72 C for 30 seconds, after initial denaturation (4 minutes at 94 C). As a control of messenger RNA quality, -actin expression was assayed with RT-PCR primers and methods detailed by Steuerwald et al. (16), quantifying transcript number in a real-time fluorescent PCR machine (Roche LightCycler; Roche Applied Sciences, Indianapolis, IN). Fertility and Sterility 1329

3 TABLE 1 Summary of results on days 6 and 12. Embryos with day-6 and day-12 results 7 Embryos with day-6 and without day-12 results 16 Embryos without day-6 and with day-12 results 16 Embryos without day-6 and without day-12 results 16 Total 55 Comparison of day-3 and day-6 results Corrected 15 Not corrected 6 Error 2 (8.7%) Total 23 Comparison of day-3 and day-12 results Corrected 9 Not corrected 6 Undetermined a 7 Error 1 Total 23 Comparison of day-3, day-6, and day-12 results Corrected 5 Not corrected 1 Error 1 Total 7 a To differentiate between a 7% 10% error rate based on one-cell analysis and true mosaic normal/ aneuploid embryos, only embryos with at least 20% abnormal cells were included in this category. Munné. Stem cells from self-corrected abnormal embryos. Fertil Steril In addition to Oct-4, hescs were also tested for SSEA-3, SSEA-4, TRA 1-60, and TRA 1-80 protein expression by immunocytochemistry with a human stem cell marker kit (Chemicon, Temecula, CA), with the TE06 hesc line (Technion, Haifa, Israel) as a positive control. RESULTS A total of 43 cycles of PGD for aneuploidy provided embryos for this study that were either diagnosed as trisomic or in a few cases had other abnormalities. In total, 142 chromosomally abnormal embryos were cultured for 2 to 3 more days, of which 55 developed into blastocyst. Because not all embryos had trophectoderm biopsy, and only 23 of the 55 embryos attached to the feeder cells, there were four types of embryos, those with FISH results on day 6 and 12 (n 7), with results on day 6 but not 12 (n 16), with results only on day 12 (n 16), and without results on either day (n 16) (Table 1). Chromosome Normalization Chromosome normalization could already be seen from day 3 to day 6. Of the 23 embryos with day-6 reanalysis, 15 showed a disomic line and one or more abnormal ones consistent with the PGD diagnosis, whereas 6 did not correct, and 2 (8.7%) were classified as PGD errors. Another set of 23 embryos had results on day 12, of which 7 had also results on day 6. Of those 23 embryos, 9 had chromosome normalization and showed a disomic line and one or more abnormal ones consistent with the PGD diagnosis, whereas 6 were still abnormal; the rest had 20% abnormal cells, thus it could not be determined whether they were PGD errors, as per our criterion described above. The embryos that showed cell normalization and had FISH analysis on days 6 and 12 are further described in Table 2. Four of the embryos (Table 2, embryos 1 4) showed a progression of normal cells, from 0 to 28% on day 6 to 21% 80% on day 12. A fifth embryo (Table 2, embryo 5) classified on day 3 as trisomy 21 did not show any trisomic cells on day 6 but was still 35% abnormal and on day 12 was 24% abnormal. A summary of all embryos with at least results on day 6 or day 12 is presented in Table 3. Confirmation of Stem Cell Production Unfortunately, of the 10 colonies assigned to be tested for Oct-4, 9 were spoiled by a power failure in our facilities. Analysis by RT-PCR showed the expression of Oct-4 by isolated fragments from the remaining colony. The PCR product s identity was confirmed by sequencing. Analysis of cellular phenotype for hesc markers showed immunepositive expression of SSEA-3, SSEA-4, TRA 1-60, and TRA DISCUSSION These data present the first evidence for normalization of chromosomally abnormal embryos during the process of hesc derivation on feeder cultures. We propose that the use of nonviable abnormal embryos is an alternative way to produce hescs, which circumvents some of the scientific and ethical restraints. It also alleviates the need for donation of potentially viable embryos in countries where egg donation might be limited owing to certain legal or financial constraints. Another proposal to develop potentially normal stem cells from abnormally developing or arrested embryos was put forth by our laboratory 5 years ago (17). That procedure involved the formation of blastocysts from the aggregation of mononucleate cells from cohorts of embryos. In the current alternative method, single embryos are used that have been analyzed for chromosome disorders Munné et al. Stem cells from self-corrected abnormal embryos Vol. 84, No. 5, November 2005

4 TABLE 2 Chromosome abnormalities found on the 3rd, 6th and 12th day of culture in embryos showing chromosome normalization. Embryo PGD day 3 TFE day 6 Day 12 1 n 1 n 54 n 117 Trisomy 13 74% trisomy 13 74% trisomy 13 26% polyploid 23% normal 0% normal 3% chaotic 2 n 1 n 37 n 92 Trisomy 15 and monosomy 22 57% monosomy 22 56% monosomy 22 24% polyploid 14% aneuploidy 15 8% aneuploidy 15 40% monosomy 22 & polyploid 11% chaotic 9% chaotic 0% normal 21% normal 3 n 1 n 18 n 85 Haploid 50% polyploid 80% normal 22% normal 7% polyploid 28% chaotic 13% monosomy 15 4 n 1 n 25 n 95 Monosomy 16 and 21 28% normal 67% normal 24% aneuploidy 16 & 21 15% polyploid 24% polyploid 12% aneuploidy 16 & 22 24% chaotic 6% chaotic 5 n 1 n 50 n 34 Trisomy 21 35% chaotic 76% normal 65% normal 24% chaotic Note: n number of cells analyzed, aneuploidy (trisomy and/or monosomy); TFE Trophectoderm. Munné. Stem cells from self-corrected abnormal embryos. Fertil Steril Demonstration of Cell Normalization It is well known that culture conditions might affect the chromosomal stability of cell lines. For instance, the fusion of mouse and human cells might produce hybrids that are known to lose human chromosomes at random until a stable form is reached. The cleavage stage is particularly prone to mosaicism in humans and certain mouse strains (18, 19). Here we demonstrate that embryonic cells derived from nonviable, chromosomally abnormal embryos can be a source of chromosomally normal cells that could be used to produce ESCs. This is exciting for a number of reasons, both scientific and ethical. The observed reduction in trisomic cells cannot be due to the nonsurvival of trisomic embryos in culture, because they survive to day 6 in sizable numbers when cultured as full embryos rather than as monolayer (20). Furthermore, the frequency of normal cells increases from day 6 (average of 13%) to day 12 (average of 48%). The most reasonable explanation for these observations is that trisomic cells selfcorrect before extended culture, yet this would need to be proven in subsequent experiments. As a method to obtain disomic stem cells, the yield is not low, with 9 of 23 chromosomally abnormal blastocysts showing disomic cells on day 12. In countries like the United States there are many constituencies that pose ethical limits on the use of human embryos other than for the purpose of conception. Obtaining stem cells from nonviable embryos might be a more ethical means of obtaining ESCs. Similarly interesting is the observation that cell normalization did not occur in some chromosomally abnormal blastocysts. Stem cell lines that have specific chromosome abnormalities could be used to study the effect of these abnormalities on human embryogenesis and for the development of therapies for aneuploidies compatible with life. Mechanism of Self-Correction It has been speculated that trisomic embryos can be corrected through three different pathways: by anaphase-lag, nondisjunction, or chromosome demolition. Anaphase-lag correction will result in one disomic and one trisomic daughter cell (21). Nondisjunction correction will result in one viable disomic and one lethal tetrasomic cell. In this circumstance, the number of cells will reduce, delaying normal development (22). The third possibility is chromosome demolition correction, proposed by Los et al. (8), which consists Fertility and Sterility 1331

5 TABLE 3 Summary of all embryos with day-6 and/or day-12 FISH results. PGD diagnosis Diagnosis day 6 Diagnosis day 12 Correction? M16 and M21 24% M16 and M21, 28% norm, 12% aneu 16 and 21, 67% norm, 24% pol, 24% chaotic 15% pol, 6% chaotic T15 and M22 57% M22, 8% aneu 15, 24% 56% M22, 14% aneu 15, 9% pol, 11% chaotic chaotic, 21% norm T13 74% T13, 26% pol 74% T13, 23% norm, 3% chaotic T21 35% chaotic, 65% norm 76% norm, 24% chaotic Complex 50% pol, 22% norm, 28% 80% norm, 13% M15, 7% pol chaotic Complex 37% MX, 37% chaotic, 26% pol 43% MX, 14% pol, 43% chaotic No M17 91% norm, 6% pol 87% norm, 13% pol Error Complex 100% chaotic No data No M 17 59% norm, 23% pol, 10% aneu No data Error 17, 5% chaotic T17 and T22 60% T17 and T22; 40% T22 No data No T17 59% T17, 12% norm, 6% pol, No data 6% hapl, 6% chaotic T21 50% T21, 50% norm No data M16 41% M16, 59% norm No data M21 25% M21, 38% norm, 37% No data M22, M18 46% M15 and M18, 46% M15, No data No 8% chaotic T13 65% T13, 25% norm, 10% pol No data T15 60% T15, 20% norm, 20% pol No data T15 79% T15, 12% norm, 4% pol, No data 4% hapl T17 45% T17, 46% norm, 9% No data triploid T22 85% T22, 15% norm No data M15 58% M15, 42% norm No data M15 and M18 41% M15, 59% norm No data M17 58% M17, 39% norm, 3% hapl No data T22 No data 13% T22, 71% norm, 16% chaotic Undetermined T15 No data 17% T15, 63% norm, 20% chaotic Undetermined T21 No data 17% T21, 75% norm, 8% chaotic Undetermined M15 and M21 No data 24% M15, 31% norm, 45% chaotic M17 No data 26% M17, 45% norm, 30% chaotic Complex No data 43% chaotic, 57% norm Complex No data 54% chaotic, 46% norm T13 and M18 No data 6% T13, 75% norm, 17% chaotic Undetermined T21 No data 69% T21, 25% chaotic, 6% norm No T22 No data 75% T22, 25% chaotic No Haploid No data 84% norm, 9% triploid, 6% chaotic Undetermined Complex No data 87% chaotic, 3% norm No T18 No data 9% T18, 82% norm, 9% chaotic Undetermined T13 No data 90% chaotic, 4% norm, 3% M13, No 3% T21 T22 No data 92% norm, 5% chaotic, 3% T22 Undetermined Complex No data 94% T22, 6% norm No Note: aneu aneuploid; hapl haploid; M monosomy; norm normal; pol polyploidy; T trisomy. Munné. Stem cells from self-corrected abnormal embryos. Fertil Steril Munné et al. Stem cells from self-corrected abnormal embryos Vol. 84, No. 5, November 2005

6 of deliberate fragmentation of one of the three chromosomes during metaphase or anaphase, resulting in two disomic daughter cells. Confined placental mosaicism, in which placental tissue showed complete trisomy, whereas the fetus was diploid, has been widely reported (23 25) asa result of the loss of trisomic chromosomes in the embryonic tissue. Once the embryo is mosaic with disomic cells, those might develop differently than the abnormal ones, and differences in cleavage performance between disomic and aneuploid cells might result in enrichment in disomic cells. The enrichment occurred, at least in part, during development from cleavage to blastocyst stage. The observation of a progression of normal cells from 12.5% on day 6 to 47.8% on day 12 in the four embryos that were diagnosed on day 6 supports the major role of post blastocyst-stage normalization. Normal diploid ESCs proliferate more slowly than karyotypically abnormal cells (26), and still the percentage of normal cells increased in these four embryos from day 6 to 12; this further supports the assumption that considerable normalization occurred before plating. The present method might be applied to obtain chromosomally normal stem cells from trisomic embryos. Because most trisomic embryos do not survive to term, those incompatible with life (e.g., trisomy 22, 15, or 16) could be a more acceptable source of chromosomally normal stem cells than normal embryos for constituencies that are now in a conundrum. Production of Disomic Stem Cells from Trisomic Embryos A fundamental property of ESCs that is routinely used in their characterization is the expression of Oct-4 transcripts. Oct-4 is a transcription factor that activates or inhibits a host of genes and maintains ESCs in a proliferating, nondifferentiating state (27). Indeed, it is believed that Oct-4 might be the master regulator responsible for preventing the expression of genes required for differentiation. Oct-4 expression seems to be tightly regulated, as evidenced by experiments in which artificially induced over- or underproduction of this protein results in divergent developmental programs (28). We have established that the cells that were derived from chromosomally abnormal embryos express this critical molecular marker of pluripotent cells. Taken together with the demonstration of the reintroduction of normal (diploid) cells, our results suggest that these cells can be used to obtain pluripotent ESCs. As such, this approach might provide a viable alternative to the derivation of stem cells from normal embryos. REFERENCES 1. Pera MF, Reubinoff B, Trouson A. Human embryonic stem cells. J Cell Sci 2000;113: Thompson JA, Odorico JS. Human embryonic stem cells and embryonic germ cell lines. Trends Biotechnol 2000;18: Thomson JA, Itskovitz-Eldor J, Shapiro SS, Wakniz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocyst. Science 1998;282: Reubinoff BE, Pera MF, Fong CY, Trouson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18: Kauolosek DK, Barrett I. Genomic imprinting related to prenatal diagnosis. Penat Diagn 1994;14: Ledbetter DH, Engel E. Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum Mol Genet 1995;4: Wolstenholme J. Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, 16 and 22: their incidence, likely origins, and mechanisms for cell lineage compartmentalization. Prenat Diagn 1996;16: Los FJ, Van Opstal D, van den Berg C, Braat APG, Verhoef S, Wesby-van Swaay E, et al. Uniparental disomy with and without confined placental mosaicism: a model for trisomic zygote rescue. Prenat Diagn 1998;18: Munné S, Sandalinas M, Velilla E, Nagy P. Polar body biopsy, embryo biopsy and fixation for FISH analysis. In: Patrizio P, Guelman V, Tucker M, eds. A color atlas of human assisted reproduction: clinical and laboratory insights. Philadelphia: Lippincott, William & Wilkins, 2003; Velilla E, Escudero T, Munné S. Blastomere fixation techniques and risk of misdiagnosis for PGD of aneuploidy. Reprod Biomed Online 2002;4: Munné S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003;7: Munné S, Magli C, Bahçe M, Fung J, Legator M, Morrison L, et al. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenatal Diagn 1998;18: Colls P, Sandalinas M, Pagidas K, Munné S. PGD analysis for aneuploidy in a patient heterozygous for a polymorphism of chromosome 16 (16qh-). Prenat Diagn 2004;24: Veiga A, Gil Y, Boada M, Carrera M, Vidal F, Boiso I, et al. Confirmation of diagnosis in preimplantation genetic diagnosis (PGD) through blastocyst culture: preliminary experience. Prenat Diagn 1999;19: Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotech 2001;19: Steuerwald N, Cohen J, Herrera RJ, Brenner CA. Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol Hum Reprod 1999;5: Alikani M, Sadowy S, Cohen J. Human embryo morphology and developmental capacity. In: Van Soom A, Boerjan M, eds. Mammalian embryo quality; invasive and non-invasive techniques. Dordrecht, The Netherlands: Kluwer Academic Publishers, Bean CJ, Hunt PA, Millie EA, Hassold TJ. Analysis of a malsegregating mouse Y chromosome: evidence that the earliest cleavage divisions of the mammalian embryo are non-disjunction-prone. Hum Mol Genet 2001;10: Munné S, Sandalinas M, Escudero T, Marquez C, Cohen J. Chromosome mosaicism in cleavage stage human embryos: evidence of a maternal age effect. Reprod Biomed Online 2002;4: Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munné S. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001;16: Kalousek DK, Howard-Peebles PN, Olson SB, Barret IJ, Dorfmann A, Black SH, et al. Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism. Prenat Diagn 1991;11: Tarin JJ, Conaghan J, Winston RML, Hanyside AH. Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage. Fertil Steril 1992;5: Fertility and Sterility 1333

7 23. Kalousek DK, Langlois S, Barrett IJ, Yam I, Wilson DR, Howard- Peebles PN, et al. Uniparental disomy for chromosomes 16 in humans. Am J Hum Genet 1993;52: Ledbetter DH, Zachary JM, Simpson JL, Golbus MS, Pergament E, Jackson L, et al. Cytogenetics results from the US collaborative study on CVS. Prenat Diagn 1992;12: Verjalav LO, Mikkelson M. European collaborative study on mosaicism in chorionic villus sampling: data from Prenat Diagn 1989;9: Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350: Pesce M, Wang X, Wolgemuth DJ, Scholer H. Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 1998;71: Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24: Munné et al. Stem cells from self-corrected abnormal embryos Vol. 84, No. 5, November 2005