1 SISMER, Reproductive Medicine Unit, Via Mazzini, 12, 40138, Bologna, Italy, 2 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, 2731 Wright Street, Clayton, Victoria, 3 Monash IVF, Monash Private Surgical Hospital, Clayton Road, Clayton, Victoria and 4 Monash IVF Gold Coast, Allamanda Medical Centre, Southport, Queensland, Australia
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: aneuploidy/blastocyst/chromosomal mosaicism/embryo biopsy/inner cell mass cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study was carried out with the aim of verifying the chromosomal status of the cells forming the ICM in blastocysts resulting from embryos identified as aneuploid at the 69-cell stage on the third day (day 3) after insemination. Blastocysts were obtained from patients, the majority of whom had a poor prognosis for pregnancy which has been demonstrated to yield 5075% chromosomally abnormal embryos (Gianaroli et al., 1997). Following preimplantation genetic diagnosis (PGD) of aneuploidy on day 3 for chromosomes X, Y, 13, 16, 18 and 21, the resulting euploid, morphologically normal blastocysts, were either transferred or cryopreserved, whereas those blastocysts classified as chromosomally abnormal were reanalysed by FISH after immunosurgical isolation of the ICM. Data expected from the chromosomal analysis using a six chromosome panel of FISH probes were: (i) to assess the rate of mosaicism in the ICM; and (ii) to define the correspondence of ICM genotype with single cell diagnosis of aneuploid 69-cell embryos.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fertilization and embryo development assessment
Oocytes were checked at 1418 h after insemination for the presence of pronuclei and polar bodies. Fertilized oocytes with two pronuclei were cultured individually and scored 48 h later for the number and appearance of nuclei and blastomeres, and the degree of fragmentation was recorded (Alikani et al., 1999). Day 3 embryos with >5 cells and
40% of fragmented cells were selected for embryo biopsy. After blastomere removal, embryos were transferred to G2 medium (Barnes et al., 1995
) and cultured for two additional days. On the morning of day 5, embryos were scored and transferred to freshly prepared G2 medium. Further observations were performed the following day to evaluate blastocyst formation and morphology (Jones et al., 1998a
).
Blastomere biopsy and FISH
Day 3 embryos selected for FISH analysis were manipulated individually in HEPES-buffered medium (Scandinavian IVF Science) overlaid with pre-equilibrated mineral oil. A breach of 2022 µm was opened in the zona pellucida with acidic Tyrode's solution and a nucleated blastomere was gently aspirated by a polished glass needle (40 µm diameter). The biopsied embryo was carefully washed and returned to culture. The removed cell was left in the micromanipulation dish at room temperature. When the biopsy procedure was completed for all the embryos, the removed blastomeres were transferred to a hypotonic solution (1% sodium citrate), the nuclei were fixed on a glass slide using methanolacetic acid 3:1, and dehydrated sequentially in 70%, 85% and 100% ethanol. Six DNA probes were used for the simultaneous detection of chromosomes X, Y, 13, 16, 18 and 21 which were labelled as follows: chromosome X with Spectrum Aqua, chromosome Y with Spectrum Aqua and Spectrum Green, chromosome 13 with Spectrum Orange and Spectrum Green, chromosome 16 with Spectrum Green, chromosome 18 with Spectrum Aqua and Spectrum Orange, and chromosome 21 with Spectrum Orange. Ten microlitres of the hybridization solution was applied to the fixed nuclei, denatured for 5 min at 73°C and left to hybridize for 4 h at 37°C in a moist chamber. After washing in 0.4xsaline sodium citrate at 71°C for 2 min, DAPI (4'6-diamidino-2-phenylindole) in antifade solution was added and fluorescence was evaluated with a Leica microscope (Leitz, Wetzlar, Germany) at x60 magnification, equipped with a triple band pass filter for the simultaneous observation of the Spectum Aqua, Spectrum Orange, and Spectrum Green signals. The X chromosome-specific signal appeared as blue, Y as whiteyellow, 13 as orange, 16 as green, 18 as pink, and 21 as red.
Blastocyst immunosurgery
All the embryos which were diagnosed as chromosomally abnormal were cultured to day 5 or 6 to form blastocysts. Following the removal of the zona pellucida with 0.2% pronase (Sigma, St Louis, MO, USA) the ICM was isolated from fully expanded and hatching blastocysts by lysing the TE cells using a complement mediated reaction. Briefly, blastocysts were individually exposed to integrin ß3 (N-20) affinity-purified goat polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at room temperature. After extensive washings, the blastocysts were transferred to 10 µl droplets of baby rabbit complement-(Serotec Ltd, Oxford, UK) for 60120 min at 4°C. The ICM were separated from the lysed TE cells and transferred to Ca2+Mg2+-free medium (Scandinavian IVF Science AB). Cell spreading was accomplished by gentle pipetting with a polished glass capillary in hypotonic solution. The separated cells were fixed on a glass slide with methanolacetic acid (3:1). Dehydration and FISH were performed as previously described.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the 16 aneuploid embryos that developed to expanded and hatching blastocysts on day 6, 15 were treated by immunosurgery. Another two blastocysts that developed from non-biopsied embryos (and were <6-cell and fragmented on day 3) and one euploid embryo were treated by immunosurgery and the ICM hybridized to FISH probes. On day 3, four embryos were trisomic, five were monosomic, one was haploid, five were complex (numerous abnormalities) and one was euploid. Two other embryos were not biopsied on day 3. The total number of cells analysed in the 18 blastocysts was 303 (16.8 ± 13.5 for each blastocyst). Fourteen blastocysts had five or more cells analysed (20.9 ± 12.5 for each blastocyst).
Table I summarizes the data obtained from FISH analysis of ICM cells of each blastocyst in relation to the diagnosis on day 3 and morphological evaluation on day 6. In all, 303 cells were examined by FISH with an average of 16.8 cells per embryo and a range of 146. In 14 embryos, the number of cells with a chromosomal diagnosis was at
5. In the remaining four embryos, the low number of cells analysed reflected either the presence of a reduced ICM or failure to screen clearly the fluorescent signals, mainly due to cell clumping. No correlation was established between embryo morphology, chromosomal status, and number of cells that constituted the ICM.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FISH analysis of interphase nuclei obtained from morphologically normal blastocysts have demonstrated that the transition from the morula to the blastocyst stage is critical in terms of starting a negative selection against aneuploid cells (Evsikov and Verlinsky, 1998). According to these authors, the degree of mosaicism observed up until the morula stage is dramatically increased in comparison to that for blastocysts. At this point, a mechanism of self-destruction is postulated to be triggered in concomitance with the first sign of clear functional polarity in the human embryo, when an evident dorsoventral symmetry arises in the blastocyst between TE and ICM cells. On the other hand, studies on 2-cell embryos have reported that TE and ICM lineages diverge very early in cleavage and mosaicism could reflect this polarity (Edwards and Beard, 1997
).
The detection of major chromosomal abnormalities, including mosaicism, in in-vitro produced blastocysts has shown that an aberrant genome is not incompatible with blastocyst development. The results reported in the present study showed 34% of euploid embryos developed to blastocysts compared to 22% for aneuploid embryos. It is apparent from these observations that long-term cultures do not exclusively select embryos with a normal chromosomal complement.
Hence in an unselected population of human blastocysts it could be expected that ~40% (as in the present study) could be chromosomally abnormal. Some of these embryos may develop to term (e.g. trisomy 21) but the majority will be lost prior to, or after implantation. The presence of blastocysts with severe chromosomal abnormalities will limit implantation and pregnancy rate even with blastocyst transfers. Claims of very high implantation and pregnancy rates after blastocyst transfer, without PGD for aneuploidy, need to be treated with caution because they might represent very highly selected patient subgroups where aneuploidy is probably very low (e.g. young women with male factor partners).
After immunosurgery and FISH on ICM cells, the numerical status of chromosomes X, Y, 13, 16, 18 and 21 was assessed in 303 ICM cells from 18 blastocysts. Multiple cell lines were demonstrated in 17 out of the 18 screened ICM. In blastocysts nos. 1, 2, and 5, the degree of mosaicism was very low (3, 6 and 9% respectively) and are probably of minor implication; monosomy was detected in two cells out of 48 (embryos nos. 1 and 2, one cell each), and monosomy and haploidy in two cells out of 22 (embryo no. 5). Similarly, in embryo no. 17, six cells out of 29 exhibited four different types of chromosomal numerical constitution. In four cases, it was due to an extra missing signal, one cell was euploid and the remaining cell was haploid. If we consider that the monosomies detected could be due to loss of micronuclei, the rate of mosaicism is possibly lower.
In the other 11 blastocysts (nos. 16 and 18 are not considered because of the small number of cells analysed by FISH: one and two respectively) the incidence of mosaicism was substantially increased. The highest values were observed in embryos nos. 8, 10 and 14, where all the screened cells appeared to be part of chaotic mosaicism. It must be considered, however, that only five, three and four cells could be diagnosed for each ICM respectively. Therefore, although present, the rate of mosaicism in these embryos could actually be similar to that detected in the remaining blastocysts. More reliable diagnoses were obtained from embryos 11, 13 and 15 which exhibited seven, 16 and 14 different cell lineages in each, out of 33, 33 and 25 screened cells respectively. This clearly demonstrates that an aberrant genome is not detrimental for blastocyst expansion and hatching.
Especially interesting is the observation of the presence of euploid cells in mosaic ICM (Table II). If mosaicism is a natural event that occurs at a low frequency in physiologically normal embryos, the reverse could also be true and result in the development of a normal fetus from the lineage of chromosomally normal cells in a mosaic embryo. Although speculative, this could possibly be related to several factors. Among them, the ratio of euploid to aneuploid cells may need to be below a hypothetical threshold. In this respect, embryo no. 6 represents an intriguing example not only in consideration of its potential development if transferred but also in relation to the reliability inherent to PGD performed on a single cell. This embryo was classified by PGD as monosomic for chromosome 18. The analysis of the ICM cells revealed 24 euploid and 22 monosomic cells. The two cell lines may originate at the first mitosis, and the chances of biopsying on day 3 a normal cell or an abnormal cell would be the same. If two cells had been removed for PGD and the result had been discrepant, the diagnosis of monosomy would probably be considered an error related to the whole procedure (loss of micronuclei during fixation or partially failed hybridization). Similarly embryo no. 13 had 14 normal out of the 33 FISH cells diagnosed. However, in this case the presence of multiple cell lineages was associated with slow cleavage rate to day 3 and probably identified a limited developmental potentiality.
According to the results of the present study, chromosomal analysis of the ICM cells of aneuploid embryos suggests that there is no limit imposed in preimplantation development by the chromosomal constitution for blastocyst formation, expansion and hatching, even in cases where a high degree of mosaicism is present. More data on ICM and trophectoderm cells are necessary to validate this hypothesis. This study has also shown a high concordance of PGD for day 3 embryos and blastocyst genotype. This affirms that the error rate due to the procedure itself (calculated to be ~8%; Gianaroli et al., 1999) is indeed low and the type of chromosomal pathology diagnosed at day 3 usually represents the onset of different cell lineages that can be identified in the ICM. In general, the correspondence between the result obtained by PGD at day 3 decreases with an increase of mosaicism in the ICM. It is also important to recognize that multiple FISH probes for the simultaneous screening of six chromosomes enables a more accurate definition of the embryo genotype. As a result, the embryos classified as carriers of complex abnormalities were all confirmed to be mosaic. Trisomies and monosomies were also confirmed with the exception of two monosomies (embryos nos. 6 and 7) where two clearly defined cell lineages, equivalent in size, developed in each embryo (one euploid and the other monosomic). There is no doubt that an increase in the number of chromosomes screened for PGD will more accurately identify the embryonic genotype in human blastocysts.
In conclusion, the chromosomal condition of the cells forming the ICM has been shown to be more heterogeneous than may have been anticipated. Further research is needed for understanding the regulatory mechanisms which determine the allocation of cell lineages within the embryo and trigger self-selection towards embryonic death or survival.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnes, F., Crombie, A., Gardner, D. et al. (1995) Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching: a case report. Hum. Reprod., 10, 32433247.[Abstract]
Bolton, V.N., Hawes, S.M., Taylor, C.T. et al. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst. J. In Vitro Fertil. Embryo Transfer, 6, 3035.[ISI][Medline]
Burgoyne, P.S., Holland, K. and Stephens, R. (1991) Incidence of numerical chromosome abnormalities in human pregnancy estimated from induced and spontaneous abortion data. Hum. Reprod., 6, 555565.[Abstract]
Delhanty, J.D.A. and Handyside, A.H. (1995) The origin of genetic defects in the human and their detection in preimplantation embryos. Hum. Reprod. Update, 1, 201215.[ISI][Medline]
Dorkras, A., Sargent, I.L. and Barlow, D.H. (1993) Human blastocyst grading: an indicator of developmental potential? Hum. Reprod., 8, 21192127.[Abstract]
Edwards, R.G. and Beard, H.K. (1997) Oocyte polarity and cell determination in early mammalian embryo. Mol. Hum. Reprod., 3, 863905.[Abstract]
Evsikov, S. and Verlinsky, Y. (1998) Mosaicism in the inner cell mass of human blastocysts. Hum. Reprod., 13, 31513155.[Abstract]
Gianaroli, L., Fiorentino, A., Magli, M.C. et al. (1996) Prolonged spermoocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum. Reprod., 11, 25072511.[Abstract]
Gianaroli, L., Magli, M.C., Munné, S. et al. (1997) Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod., 12, 17621767.[Abstract]
Gianaroli, L., Magli, M.C., Munné, S. et al. (1999) Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J. Assist. Reprod. Genet., 16, 170175.[ISI][Medline]
Hardy, K. (1994) Effects of culture conditions on early embryonic development. Hum. Reprod., 9, 9499.[Abstract]
Hardy, K., Handyside, A. and Winston, R.M.L. (1989) The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development, 107, 597604.[Abstract]
Janny, L. and Ménézo, Y.J.R. (1996) Maternal age effect on early human embryonic development and blastocyst formation. Mol. Reprod. Dev., 45, 3137.[ISI][Medline]
Jones, G.M., Trounson, A.O., Lolatgis et al. (1998a) Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer. Fertil. Steril., 70, 10221029.[ISI][Medline]
Jones, G.M.., Trounson, A.O., Gardner, D.K. et al. (1998b) Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum. Reprod., 13, 169177.[Abstract]
Kaufman, R.A., Ménézo, Y., Hazout, A. et al. (1995) Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil. Steril., 64, 11251129.[ISI][Medline]
Magli, M.C., Gianaroli, L., Munné, S. et al. (1998) Incidence of chromosomal abnormalities in a morphologically normal cohort of embryos in poor-prognosis patients. J. Assist. Reprod. Genet., 15, 296300.
Magnuson, T., Debrot, S., Dimpfl, J. et al. (1985) The early lethality of autosomal monosomy in the mouse. J. Exp. Zool., 236, 353360.[ISI][Medline]
Munné, S., Alikani, M., Tomkin, G. et al. (1995) Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil. Steril., 4, 382391.
Plachot, M., Veiga, A., Montagut, J. et al. (1988) Are clinical and biological IVF parameters correlated with chromosomal disorders in early life? A multicentric study. Hum. Reprod., 3, 627635.[Abstract]
Trounson, A. (1983) Factors controlling normal embryo development and implantation of human oocytes fertilized in vitro. In Beier, H.M and Linder, H.R. (eds), Fertilization of the Human Egg In Vitro. Springer-Verlag, Berlin, pp. 235225.
Submitted on November 22, 1999; accepted on May 4, 2000.