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Embryo |
a Department of Reproductive Science and Medicine, Institute of Reproductive and Developmental Biology, The Wolfson and Weston Research Centre for Family Health, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom
b Department of Mathematics, University College London, London WC1E 6BT, United Kingdom
c CoMPLEX (Centre for Mathematics and Physics in the Life Sciences and Experimental Biology), University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, early development, embryo, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Little is known about the effects of fragmentation on blastocyst cell number, apoptosis, or the allocation of cells to the inner cell mass (ICM), from which the fetus is derived, and the trophectoderm (TE). The increasing use of blastocyst culture in clinical IVF and selection of blastocysts for transfer has stimulated this retrospective analysis of data that we collected over the past decade on blastocyst formation, cell number, and cell death. Focusing on embryo development in a single culture medium, we examined the impact of fragmentation during early cleavage on subsequent blastocyst formation and cell number. We also assessed the number of cells allocated to the ICM and the TE and the incidence of apoptosis in these two lineages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human Oocytes and Embryos
Embryos were donated, with informed consent, by infertile couples undergoing IVF who did not wish to cryopreserve untransferred embryos for future replacement. The work was licensed by the Human Fertilisation and Embryology Authority, and local permission was granted by the ethics committee of the Royal Postgraduate Medical School, Hammersmith Hospital, London.
Superovulation in women undergoing IVF treatment was induced with human menopausal gonadotropin (Pergonal; Serono, Feltham, Middlesex, U.K.) after pituitary-gonadal suppression by an LH-releasing hormone agonist (Buserelin; Hoescht, Hounslow, U.K.) [12]. Ten thousand international units of hCG (Profasi; Serono) was administered 36 h before oocyte retrieval. Oocyte-cumulus complexes were preincubated, inseminated, and observed the following day for the presence of pronuclei [13]. Between the time when embryos were checked for pronuclei, until the time of embryo transfer, embryos were cultured singly in 1 ml of EBSS (Gibco BRL, Paisley, U.K.) containing 5.56 mM glucose, 25 mM sodium bicarbonate (BDH, Lutterworth, Leics, U.K.), 0.47 mM pyruvate (Sigma, Poole, Dorset, U.K.), 37.5 U/ml of streptomycin (Sigma), and 97.5 U/ml penicillin (Sigma) and supplemented with 10% heat-inactivated maternal serum under a gas phase of 5% CO2, 5% O2, and 90% N2 at 37°C.
Classification and Grading of Embryos
On the morning of Day 1, approximately 16–18 h postinsemination (Day 0 = day of oocyte retrieval and insemination), oocytes were classified according to the number of visible pronuclei. On the morning of Day 2, embryos were graded on the basis of blastomere symmetry and degree of fragmentation (Fig. 1). Grade A embryos had symmetrical or very slightly asymmetrical blastomeres with either no fragmentation or occasional small fragments (<5%); grade B embryos had all the blastomeres intact but some cytoplasmic fragmentation (5–10%) and/or unevenly sized cells; grade C embryos had asymmetry and more extensive fragmentation (10–25%) although all the blastomeres remained intact; and grade D embryos had one or more fragmented blastomeres (>25% fragmentation). The number of blastomeres was also assessed.
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One to three of the most rapidly developing embryos with the closest to perfect morphology were selected for transfer [14]. Untransferred embryos were cryopreserved or donated, with informed consent, for research. All embryos included in this analysis had two pronuclei on Day 1, with no evidence of polyspermic fertilization.
Culture of Embryos
From Day 2 on, after embryo transfer surplus embryos were individually cultured in 5-µl drops of custom-made EBSS with 1 mM glucose and supplemented with 2.5% HSA in filter-sterilized silicone fluid (Dow Corning 200/50 cs; BDH) under a gas phase of 5% CO2 in air. Embryos were moved to fresh drops daily. Embryos were scored between 1200 and 1700 h on Day 2.
Counting of Blastocyst Nuclei
On the morning of Day 6, blastocysts were scored for the degree of blastocoele expansion. The cavities in early blastocysts and blastocysts were less than half and greater than half the diameter of the embryo, respectively. When the diameter of the blastocysts had markedly increased (
20%), with zona thinning, the embryo was classified as an expanded blastocyst. Cell counts for the TE and ICM of the blastocysts were obtained by differential labeling of the nuclei with polynucleotide-specific fluorochromes [15]. The TE nuclei were labelled with propidium iodide during immunosurgical lysis, before fixing the embryo and labeling both sets of nuclei with bisbenzimide in absolute ethanol. Differentially labeled embryos were mounted in glycerol and partially disaggregated, and the nuclei were counted under fluorescence microscopy. When viewed through a combination ultraviolet excitation filter and fluorescein isothiocyanate emission filter, the TE and ICM nuclei appear orange and green, respectively [15]. Before disaggregation, embryos were inspected in whole mount to assure that the entire TE was uniformly labeled. In some embryos, only part of the TE was labeled with propidium iodide, probably because incomplete removal of the zona pellucida had impeded the immunosurgical lysis. For these embryos, only measurements of total cell number were considered accurate.
Apoptotic nuclei were easily identified and characterized by discrete clusters of labeled nuclear fragments. Apoptotic nuclei were not included in the total numbers of healthy nuclei in the TE and ICM. The dead cell index was computed:
Statistical Analysis
Blastocysts were grouped according to the morphological grade that they had been earlier in development, on Day 2 (grades A–D). Numbers of embryos reaching the blastocyst stage in all the groups were compared using chi-square analysis for multiple groups. When the difference was significant (P < 0.05), differences between individual pairs of groups were compared using chi-square analysis, and the P value was then corrected using the Bonferroni correction (multiplying the P value by the number of comparisons initially made, i.e., n(n - 1)/2, where n is the number of groups).
Numbers of healthy cells were normally distributed, but the populations in groups A–D had significantly different SDs (P < 0.01, using the method of Bartlett). In addition, the percentage of apoptotic cells was not normally distributed in the different groups. Therefore, differences in the distribution of the total cell number, the number of TE and ICM cells, and the percentage of apoptotic cells were compared first using the Kruskal-Wallis test (a nonparametric test for more than two groups). When the differences in distribution were significant for all the groups, individual comparisons between two groups of interest were made using a Dunn posthoc test.
Statistical analyses were performed using GraphPad InStat version 3.0a for Macintosh (GraphPad Software, San Diego, CA; www.graphpad.com).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, 200/363 (55%) of embryos reached the blastocyst stage by Day 5 or Day 6. The remaining embryos arrested at or after the four-cell stage.
Embryo grade on Day 2 affected the subsequent rate of development, significantly affecting compaction on Day 4 (P = 0.0001) and cavitation on Day 5 (P = 0.008). By Day 4, 47% of grade A, 36% of grade B, 37% of grade C, and only 9% of grade D embryos had reached the morula stage. On Day 5, 19% of grade A embryos but only 5% of grade D embryos cavitated.
Embryo grade on Day 2 was related significantly to blastocyst formation by Day 6 (P = 0.0001); only 12% of grade D embryos but >60% of grade A and grade B embryos gave rise to blastocysts (Fig. 2). Chi-square tests showed strong evidence for a trend in the proportion of embryos reaching the blastocyst stage in relation to increasing fragmentation on Day 2. Early cleavage rate has been reported as related to subsequent blastocyst formation [16, 17]. Even when only embryos with four or more cells on Day 2 were considered, there was a significant decrease (P = 0.0006) in blastocyst formation, from 62% for grade A embryos to 16% for grade D embryos.
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A similar proportion of blastocysts from embryos of grades A–C were expanded (56–61%). No expanded blastocysts developed from grade D embryos.
Blastocyst Cell Number
Embryo grade on Day 2 was also significantly related (P = 0.003) to the number of cells in Day 6 blastocysts (Fig. 3A), with cell number declining from 68.9 ± 5.5 (mean ± SEM) in grade A embryos to 29.0 ± 3.6 in grade D embryos. This reduction in cell number was mainly due to a steady and parallel decline in the number of TE cells (P = 0.002) (Fig. 3B). When only embryos with four or more cells on Day 2 were considered, there was still a significant decrease (P < 0.05) in the number of cells in Day 6 blastocysts from 74.7 ± 6.5 for grade A embryos to 31.5 ± 3.3 for grade D embryos, with a similar decline in the number of TE cells (data not shown). Although a similar proportion of blastocysts were expanded in grades A–C, the cell number in expanded blastocysts declined from 74.6 ± 6.3 (grade A) to 52.3 ± 4.6 (grade C) (nonsignificant difference).
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The decline in the number of TE cells was significant even in blastocysts where all the blastomeres had been intact on Day 2 (grades A–C; P = 0.03). The number of ICM cells remained remarkably constant (P = 0.248).
Embryo grade on Day 2 was significantly related to apoptosis in Day 6 blastocysts (P = 0.0005) (Fig. 3C). The dead cell index significantly declined from 10% in blastocysts from grade A embryos to 3% (P < 0.01) in blastocysts from grade B embryos. For both grade A and grade B embryos, levels of apoptosis were similar in the TE and ICM (Fig. 3D). Level of apoptosis in blastocysts from grade C embryos was significantly higher than that for blastocysts from grade B embryos (Fig. 3C; P < 0.05), although apoptosis in the ICM remained low (Fig. 3D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In both the present and a previous study [8], a small degree of fragmentation (up to 10–15%) did not affect blastocyst formation, but when fragmentation was >15%, the rate of blastocyst formation declined rapidly. There are several possible explanations for the potentially detrimental effects of fragments on development. Fragments may physically impede cell-cell interactions, interfering with compaction, cavitation, and blastocyst formation [4, 18]. Ultrastructural observations that blastomeres adjacent to fragments show signs of degeneration [19] have given rise to the idea that fragments release toxic substances that damage nearby cells [4]. Fragments may also reduce the volume of cytoplasm and deplete the embryos of essential organelles or polarized domains [4, 20].
During cleavage, there is no cell growth [21]. Thus, fragmentation could result in loss of whole blastomeres, loss of cytoplasm from individual blastomeres, or both. Previous studies have shown that removal of entire blastomeres (to a maximum of 25% of the embryo volume) from four- and eight-cell mouse and human embryos produces blastocysts with fewer cells. However, removal of cells has no effect on blastocyst formation [22–24]. In contrast, in the present study severe fragmentation equivalent to loss of one or more blastomeres on Day 2 was associated with a dramatically reduced blastocyst formation rate, from 60% to 12%. This finding implies that the reduction in blastocyst formation is not simply due to the decrease in embryo volume resulting from fragmentation.
This first study of the effects of fragmentation on blastocyst cell number revealed that increasing fragmentation is associated with a decrease in cell number on Day 6. Even embryos with only minor fragmentation and no loss of blastomeres have reduced cell numbers on Day 6. Because there is no cell growth during cleavage, formation of anucleate cytoplasmic fragments will result in decreased cell volume. In the mouse, reducing cell volume by removing cytoplasm from fertilized oocytes leads to a reduced cell division rate and lower blastocyst cell numbers, and extreme reduction of cell volume (to the extent that the cell is simply a karyoplast) stops cell division altogether [25–27]. This finding supports the hypothesis that increasing fragmentation (and hence reduced cell volume of intact blastomeres) in human embryos results in slower cell division and hence low cell numbers in the blastocyst. Thus, it appears that reducing the volume of the embryo by removing entire blastomeres decreases blastocyst cell number but does not affect blastocyst formation. However, reducing the volume of individual blastomeres by removing cytoplasm (either artificially by micromanipulation or naturally by fragmentation) decreases both blastocyst formation and cell number.
As overall cell numbers decrease, the two lineages show distinctly different behaviors. With declining cell numbers, the number of ICM cells is maintained at a remarkably steady level at the expense of TE cell number, which diminishes steadily with increasing fragmentation. ICM cell numbers only dropped in blastocysts that at earlier stages had gross (>25%) fragmentation. There seems to be homeostatic control of ICM cell numbers, and the mechanism for this control apparently remains intact up to moderate levels of fragmentation. The number of ICM cells could be controlled at the stage when cells are allocated to the ICM. In the mouse, allocation to the ICM takes place initially during the fourth cleavage division and is fine-tuned by a further contribution from the outer cells during the fifth division, maintaining ICM cell numbers within a fairly narrow range [28]. Alternatively, the number of ICM cells in the expanding blastocyst could be maintained by adjusting the balance between cell division and cell death, as has previously been suggested [29, 30]. Levels of cell death in the ICM are high in blastocysts from grade A embryos of perfect morphology (and high cell numbers) and are lower in embryos of poorer quality (grades B and C) with lower cell numbers (Fig. 3D), supporting a regulatory role for apoptosis in maintenance of cell number.
The hypothesis that fragmentation reflects inherent abnormalities of embryogenesis is supported by previous observations that extensive fragmentation is associated with aneuploidy, increased mosaicism, and polyploidy [31–34]. These abnormalities may result in developmental arrest and reduced cell numbers in surviving blastocysts. However, it is also possible that chromosomal abnormalities do not cause fragmentation but rather that chromosomal abnormalities and fragmentation go hand in hand, both being a manifestation of cytoskeletal and spindle defects. Mosaicism is thought to arise after fertilization, during mitosis, as a result of either a lack of cell cycle checkpoints [35] or a defective mitotic apparatus. These defects could result in asymmetric and abnormal cell division, nondisjunction, anaphase lag, and formation of micronuclei [19], which would in turn give rise to aneuploid cells [33, 36, 37], cells with nuclear abnormalities (such as bi- and multinucleate cells) [38], and anucleate cells or fragments. The manifestation of fragmentation on Day 2, before global activation of the embryonic genome [39, 40], probably reflects a fundamental defect that originated in the gametes, most likely the oocyte [41]. The increased levels of apoptosis seen in grade C and grade D embryos (Fig. 3C) could be associated with removal of defective cells from the embryo, as proposed by Hardy [42]. Alternatively, fragments may be equivalent to apoptotic bodies [43]. However, we and others (unpublished observations) [18], using classic markers such as TUNEL labeling of fragmented DNA or annexin V labeling of externalized phosphatidylserine, have been unable to confirm that fragments are the end products of apoptosis.
There is evidence that the extent of fragmentation is modulated by the environment. Embryos cultured on a monolayer of feeder cells had fewer fragments than did embryos cultured alone [44]. Efforts to improve culture media, in particular the advent of sequential media designed to support the changing nutritional requirements of the embryo, prompt the question of whether a similar association between the degree of fragmentation and developmental potential, cell number, and apoptosis operates in embryos cultured in different media.
The high level of apoptosis seen in embryos of perfect morphology is intriguing. In addition to removal of defective cells, apoptosis could be playing a homeostatic role, regulating cell number and preventing overgrowth, particularly of the ICM. An oversize ICM could have problems maintaining healthy central cells because of the increased distance over which oxygen and nutrients would have to diffuse. In addition, a disproportionately large ICM could result in an oversize fetus and large-offspring syndrome, as has previously been proposed [45]. As yet, the role of apoptosis is preimplantation development is unknown. Although the human embryo can tolerate and may even require moderate levels of apoptosis for healthy development, excessive cell death will most probably result in implantation failure or embryonic loss soon after implantation.
Fragmentation has a detrimental effect on blastocyst formation and cell number. Regardless of whether the reduced cell number is due to reduced blastomere volume following fragment formation or is a reflection of an inherent defect in the embryo (such as aneuploidy), a constant number of ICM cells is maintained. Only in excessively fragmented embryos is cell number reduced in both lineages. These results suggest that a small degree of fragmentation (up to 10–15%) is not necessarily indicative of major defects in the embryo or of a reduced capacity to form a blastocyst. Rather, limited fragmentation may merely lead to a slower rate of cell division, probably because of a loss of cytoplasmic volume. However, excessive fragmentation (>25%) is associated with very poor blastocyst development, probably reflecting inherent defects in the embryo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 7 August 2002.
First decision: 23 August 2002.
Accepted: 11 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
2 test with Bonferroni correction)
) and in ICM (
) in blastocysts. C) Percentage of apoptotic nuclei in blastocysts. Bars with the same superscript are significantly different (a, P < 0.01; b, P < 0.05). D) Percentage of apoptotic nuclei in the TE (