Hypocellularity and absence of compaction as criteria for embryonic death

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1 SPECIAL REPORT Hypocellularity and absence of compaction as criteria for embryonic death Donald W Landry 1, Howard A Zucker 2, Mark V Sauer 2, Michael Reznik 2 & Lauren Wiebe 2 Author for correspondence 1 Director, Division of Experimental Therapeutics Columbia University P&S Building, West 168 Street New York, NY 10027, USA Tel.: ; Fax: ; DWL1@ columbia.edu 2 Departments of Medicine, Pediatrics and Obstetrics & Gynecology, Columbia University, New York, NY, USA Background: A precise definition of death is important for the appropriate application of medical resources and the harvesting of tissues for transplantation. For developed humans, life is considered to end when the criteria for brain death are met, but corresponding criteria are lacking for human embryos, and thus, we undertook a natural history study of embryonic death. Methods: De-identified records of the observations of human embryos in culture were analyzed retrospectively. The embryos were generated by in vitro fertilization for the purpose of reproduction. Cell number and morphology were recorded on embryonic days 2, 3, 5, and 6. Viable embryos (n = 248) were compared to nonviable embryos (n = 444) and the latter were analyzed in subgroups defined by cell number and morphology. Results: Many nonviable embryos (n = 142 out of 444) were hypocellular and lacked compaction on embryonic day 5 (ED5). All of these hypocellular embryos did not progress to compacted morula or normal blastocyst when observed further. No criteria could be discerned for the diagnosis of death on ED3. Conclusions: Arrested development at the multicellular stage on ED5 indicates an irreversible loss of integrated organic function, and hence, the condition of death for the organism. Approximately a fifth of all embryos generated for in vitro fertilization heretofore misclassified among the nonviable are in fact dead on ED5 by our criteria. We propose that the ethical framework currently used for obtaining essential organs from deceased persons for transplantation could be applied to the harvesting of live cells from dead human embryos for the creation of stem cells. Keywords: compaction, embryonic death, hypocellularity Death, long diagnosed by an absence of cardiac or pulmonary activity or the presence of rigor mortis, was re-evaluated in the 1960s because of advances in life-supporting technology and the implications of this diagnosis for medical interventions [1]. Grounded in the premise that the life of a multicellular organism is greater than the sum of the lives of its individual cells, the modern concept of death is based on an irreversible loss of integrated organic function [2], not necrosis. This concept is applied to the death of the developed human being through the application of criteria for the diagnosis of brain death.[3]recently, we proposed to extend this concept to the death of the human being at the embryonic stage of development [4]. We suggested that for a stage preceding development of the nervous system, irreversible arrest of normal development could provide an alternative marker for the irreversible loss of integrated organic function. Criteria that would establish irreversible cessation of development (by analogy with absence of electric activity on electroencephalography) need to be noninvasive, readily interpretable and sufficiently decisive to allay anxieties regarding the possibility of reversibility. We now propose such criteria that are based on our observation of the natural history of nonviable early embryos engendered by in vitro fertilization (IVF). These criteria would provide an alternative to the current approach to embryo death based on the death of every cell. Human embryos, whether residing in utero or in vitro, have a high mortality rate, with an incidence of death well exceeding 50% during the first few days of embryonic life [5]. In vitro fertilization performed for reproductive purposes yields hundreds of thousands of embryos each year, the majority of which are deemed nonviable and discarded. An embryo s lack of viability is diagnosed through defects in form and function [6]. Morphologic criteria for nonviability include fragmentation, loss of cytoplasm and decrease in cytoplasmic clarity. The functional criterion is an arrest of normal growth and development manifested by persistent hypocellularity and immature cell types. Nonviability merely indicates the lack of a capacity to develop to live birth and does not necessarily imply death: the term nonviable subsumes dead, dying and / xxx 2006 Future Medicine Ltd ISSN Regenerative Med. (2006) 1(3), xxx xxx 371

2 SPECIAL REPORT Landry, Zucker, Sauer, Reznik & Wiebe doomed embryos. Nonetheless, as noted above, the functional criterion for nonviability arrested development might in fact correlate with criteria for death. The high mortality of early embryonic life is largely the result of genetic defects, and aneuploidy is the most common abnormality [7]. However, mosaicism is unexpectedly common and normal diploid cells can be found in up to a quarter of nonviable embryos [7,8]. In a milieu of abnormal cells, ostensibly normal cells may fail to develop further. This failure to develop is not an intrinsic defect but rather a manifestation of the collapse of integrated organic function: some of these same cells, if transplanted into a normal embryo, can be reprogrammed for continued growth and development [6,9]. Criteria for irreversible loss of the capacity for normal embryonic development would provide an operational definition for the diagnosis of embryo death consistent with the concept of death as the irreversible loss of integrated organic function. The issue of irreversibility is a paramount concern for any determination of death. Thus, we proposed [4] that a natural history study of nonviable IVF embryos might yield a time beyond which an embryo, having failed to divide normally despite best efforts, never returns to the path of normal development. Methods Procedures & participants With the approval of the Columbia University Institutional Review Board, we examined the de-identified records of human embryos generated for the purpose of reproduction at the Center for Women s Reproductive Care at Columbia University. A clinic with national standing was selected in order to identify criteria for death not skewed by a deficient quality of care. Human embryos, prepared in the usual manner [10] were inspected as is routine only once daily and only on days 2, 3 and 5 and/or 6. Normal development results in an embryo of approximately six to ten cells on the third embryonic day (ED3) and a blastocyst on ED5. Such embryos, if also deemed viable by morphologic criteria, were either transferred in uterum or frozen in anticipation of a future cryo IVF cycle. The remaining embryos, found to be morphologically defective and/or hypocellular for their embryonic age, were retained in culture. The natural history of the nonviable embryos was assessed by analyzing subgroups based on cell number and morphology. Statistical analysis Cell number for multicellular-stage embryos and viable embryos on individual embryonic days were compared using a t-test. Comparisons on ED3 between the multicellular group, the viable group, and the nonviable compacted morula and blastocyst group were made using ANOVA. Role of funding This work was funded by the 1998 Gambro Charitable Trust (DWL). This source had no influence on any aspect of the study. Results The data for embryos generated between January and June of 2005, with records of inspection on both ED5 and ED6, revealed 43 IVF cycles producing 692 embryos with two pronuclei after fertilization. A total of 248 viable embryos were transferred and/or frozen as above; 444 defective embryos were observed. Analysis of this latter group focused on those embryos found to be hypocellular on ED5 and designated by the IVF center as multicellular or morula. In this classification system, multicellular, often defined as two to six cells, [11] denotes development up to the stage of the early morula with large, distinct, countable cells numbering about nine to ten or fewer, and morula describes a mature structure of up to 30 smaller, compacted cells. The ED5 multicellular embryo was of particular interest because of its unambiguous departure from normal development: it has failed to advance to the compacted morula anticipated on ED4 and thus is far removed from the anticipated ED5 blastocyst with ~60 80 small cells and an inner cell mass the locus of further embryonic development and, historically, the source of embryonic stem cells [12]. Markedly hypocellular embryos typically degenerate and fragment beyond ED6 and thus the data were analyzed to test the hypothesis that such embryos arrested on ED5 never resume the path of normal development on ED6. We found that 142 embryos were designated multicellular on ED5 and were inspected on ED6, at which time a mature blastocyst would be expected. Only immature hypocellular embryos were observed on ED6: 84% multicellular embryos, 12% markedly abnormal embryos (e.g., with vacuolization) that show some sign of cavitation but without the inner cell mass of a normal blastocyst and 4% further degenerated embryos, e.g., with frank loss of cells and blackened areas within 372 Regenerative Med. (2006) 1(3)

3 Hypocellularity and absence of compaction as criteria for embryonic death SPECIAL REPORT cells. No embryo among the 142 progressed to the normal designations morula or blastocyst. All 142 embryos were discarded on ED6 as nonviable (Figure 1). The designation morula on ED5 also indicated a profound defect in integrated function, demonstrated by the fact that none of 28 ED5 compacted morulas developed into normal blastocysts on ED6. An effort to diagnose embryo death earlier than ED5 proved inconclusive. The average cell count on ED3 for the 142 multicellular embryos (5 61 ± 1 65) was significantly less by ANOVA than that on ED3 for viable embryos (7 63 ± 1 06; p < 0 001) and for nonviable compacted morulas and blastocysts, i.e., the dying and doomed embryos (6 57 ± 1 66; p < 0 001). However, there is considerate overlap between groups and the data does not support the possibility of a criterion for discerning death based on ED3 cell number. The remainder of Figure 1. Comparative failure of growth for ED5 multicellular embryos. Total cell count Embryonic day The average cell count for the 142 embryos identified as multicellular on ED5 (solid circles, lower curve) is compared with that of viable embryos (upper curve). The 248 embryos identified as viable are shown on ED2 and 3 (open circles); data abstracted from published sources [13,14] are depicted for ED5 and 6 (triangles +SD). All ED2 and 3 data are shown with one-sided SD bars; data for ED5 and 6 multicellular embryos are estimates (without error bars) derived from simple inspection, not disaggregation or staining (p < 0.05 for all daily comparisons). SD: Standard deviation. the 444 nonviable embryos were morphologically defective blastocysts that had not undergone unambiguous arrest by ED6 and thus were not yet dead as organisms. Discussion Based on these data, we propose that hypocellularity and lack of compaction on ED5 constitute a set of criteria for diagnosing an irreversible arrest of normal development for human embryos and, in direct correspondence to the paradigm for brain death, are sufficient criteria for the diagnosis of death. The human embryo satisfying these criteria has died as an organism even if certain individual cells remain alive (Figure 2). The adequacy of these criteria rests on this central finding: no such ED5 embryo ever developed into a normal blastocyst with an inner cell mass. Our proposed criteria will be confirmed and potentially refined through a prospective study of larger numbers of continuously observed nonviable embryos, followed to the time of complete degradation. A further refinement could entail inclusion of additional criteria for death, such as hypocellularity on ED3 and/or the presence of some degree of abnormal morphology. Our criteria must be separately validated for ED3 embryos that were originally frozen and then thawed. Longer and more detailed observation of nonviable ED5 compacted morulas and blastocysts may provide analogous criteria for embryonic death at these stages of development. Human embryonic stem cells have recently been generated from an early morula the structure that on ED5 indicates death and so a test of the developmental potential of cells donated from dead human embryos is now possible [15]. Similarly, recent advances in the generation of stem cells from a single biopsied blastomere also support further work with individual cells from dead embryos [16]. Approaches utilizing reprogramming of somatic cells through fusion with embryonic stem cells [17] would be facilitated if a ready supply of embryonic stem cells could be achieved without the destruction of human embryos. The recent report of ostensibly normal blastomeres derived from genetically defective human embryos provides further support for a program to derive stem cells from the subclass of nonviable embryos that are dead [18]. Based on our data, approximately three embryos per IVF cycle could be diagnosed as dead on ED5. Each of the several hundred IVF clinics in the USA alone undertakes several hun

B: embryo 1 with normal compaction and blasocyst formation on ED5 and embryo 2 arrested at the multicellular stage on ED5.

4 SPECIAL REPORT Landry, Zucker, Sauer, Reznik & Wiebe Figure 2. Diagnosis of death on ED5. Two embryos from the same donor couple were observed. A Embryo #2 ED5 B Embryo #1 ED3 Embryo #2 ED3 Embryo #1 ED5 A: embryos 1 and 2 at the multicellular stage on ED3. B: embryo 1 with normal compaction and blasocyst formation on ED5 and embryo 2 arrested at the multicellular stage on ED5. Embryo 2 is declared dead on ED5 through application of our criteria for embryonic death. dred cycles per year. Thus, dead IVF embryos originally engendered for reproductive purposes are potentially an abundant source of cells. In contrast to some approaches to embryonic stem cells that propose to spare human embryos [19],this approach does not require preliminary work in animal models for proof of concept. The requirement, in current practice, that human embryos be destroyed in order to create human embryonic stem cells has impeded biomedical research. For example, the ethical controversy arising from this destruction has led to a stalemate on federal funding within the USA that cannot be fully addressed by local grants and commercial investment. The additional ethical problem of obtaining human oocytes for research and development underscores the diffi- Bibliography Ad Hoc Committee of the Harvard Medical School: A definition of irreversible coma. JAMA 205, (1968). Capron A, Kass L: A statutory definition of the standards for determining human death; an appraisal and a proposal. U. Penn. Law Rev. 121, (1972). Report of the Quality Standards Subcommittee of the American Academy of culties in this area. We propose that the translation of the universally accepted ethical framework for essential organ donation to the harvesting of cells from dead human embryos could provide a common ground in which the imperative to safeguard human dignity and the drive for biomedical research are not in conflict. Contributors We declare that we participated in the design of the study (DWL, HZ, MS), the literature review and data analysis (DWL, MR, LW), and the writing of the manuscript (DWL), and that we have all seen and approved the final version. Conflict of Interest Statement None of the authors has a conflict of interest. Neurology. Practice parameters for determining brain death in adults. Neurology 45, 1912 (1995). Landry DW, Zucker HA. Embryonic death and the creation of human embryonic stem cells. J. Clin. Invest. 114, 9 (2004). Alikani M, Calderon G, Tomkin G, Garrisi J, Kokot M, Cohen J: Cleavage anomalies in early human embryos and survival after prolonged culture in vitro. Hum. Reprod. 15(12), (2000). Alikani M, Willadsen S: Human blastocysts from aggregated mononucleated cells of two or more nonviable zygote-derived embryos. Repro. Biomed. Online 5, (2002). Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative Regenerative Med. (2006) 1(3)

5 Hypocellularity and absence of compaction as criteria for embryonic death SPECIAL REPORT genomic hybridization. Hum. Genet. 106, (2000). 8. Laverge H, Van der Elst J, De Sutteer P, Verschraegen-Spae MR, De Paepe A, Dhont M: Fluorescent in situ hybridization on human embryos showing cleavage arrest after freezing and thawing. Human Reprod 13, (1998). 9. Byrne JA, Simonsson S, Gurdon JB: From intestine to muscle: Nuclear reprogramming through defective cloned embryos. Proc. Natl Acad. Sci. USA 99, (2002). 10. Peña J, Chang PL, Thornton MH, Sauer MV: Serum estradiol levels after 4 days of ovarian hyperstimulation in oocyte donors are predictive of embryo quality and clinical outcomes. Gynecol Obstet Invest 54, (2002). 11. Kattera S, Shrivastav P, Craft I: Comparison of pregnancy outcome of pronuclear- and multicellular-stage frozen-thawed embryo transfers. J. Assist. Reprod. Gen. 16(7), (1999). 12. Thomson JA, Itskovitz-Eldor, J, Shapiro SS, et al.: Embryonic stem cell lines derived from human blastocysts. Science 282, (1998). 13. Conaghan J, Hardy K, Leese HJ, Winston RM, Handyside AH: Culture of human preimplantation embryos to the blastocyst stage: A comparison of 3 media. Int. J. Dev. Biol. 42, (1998). 14. Fong C-Y, Bongso A: Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture. Hum. Reprod. 14(3), (1998). 15. Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y: Morula-derived human embryonic stem cells. Reprod. Biomed. 9(6), (2004). 16. Chung Y, Klimanskaya I, Becker S et al.: Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439(7073), (2006). 17. Cowan CA, Atienza J, Melton DA, Eggan K: Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, (2005). 18. Check E: Biologists forced to reassess embryo test. Nature 437(7062), 1075 (2005). 19. The President s Council on Bioethics. Alternative Sources Of Human Pluripotent Stem Cells: A White Paper. Washington, DC, USA (2005)

Preimplantation genetic diagnosis: polar body and embryo biopsy

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