Department of Obstetrics and Gynaecology, Göteborg University, SU/Sahlgrenska, 413 45 Gothenburg, Sweden
1 To whom correspondence should be addressed. e-mail: thorir.hardarson{at}obgyn.gu.se
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Abstract |
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Key words: blastocyst/pre-embryo/aneuploidy/FISH/IVF
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Introduction |
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It has been suggested that culture of surplus pre-embryos (i.e. pre-embryos of suboptimal quality, not transferred or frozen on day 2 or 3) to the blastocyst stage would enhance the selection of pre-embryos with high implantation potential (Balaban et al., 2001). Genetic studies of blastocysts from such surplus pre-embryos, in particular studies using suitable control groups, are limited. Several studies concerning chromosomal aberrations in the human blastocyst have been performed, mainly using the fluorescence in-situ hybridization (FISH) technique (Benkhalifa et al., 1993
; Evsikov and Verlinsky 1998
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000
; Sandalinas et al., 2001
; Bielanska et al., 2002
). These previous studies include analyses of blastocysts from a variety of patient groups but with no control groups. In contrast, this study was designed to use a clinical approach to study blastocysts, comparing, both morphologically and chromosomally, blastocysts obtained from morphologically low quality pre-embryos with blastocysts that develop from good quality embryos (GQE). In addition, our aim was to find out if improvement of the morphology and/or developmental state of pre-embryos between day 2 and day 3 is predictive of a chromosomally normal blastocyst.
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Materials and methods |
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Ovarian stimulation was carried out by a desensitizing protocol using a short-acting GnRH agonist preparation in combination with recombinant FSH. Follicular aspiration was performed 3638 h after hCG administration using vaginal ultrasonography. The oocytes either underwent ICSI or were inseminated in our conventional IVF programme, after which they were cultured in IVF-20 (Vitrolife Ltd, Gothenburg, Sweden) for two days. On the day of embryo transfer (day 2), the pre-embryos were scored according to our grading system. This system is modified from Steer et al. (1992) and consists of four main pre-embryo morphological grades, where a developmental rate of 46 cells at day 2 and
6 cells on day 3 is considered normal. Grade I is considered the optimal pre-embryo, with no fragments, even-sized blastomeres and light, homogeneous cytoplasm. Grade II is divided into three categories: <20% fragments, uneven-sized blastomeres and/or non-homogeneous cytoplasm. Remaining pre-embryos fall into the third and fourth main groups, mainly due to high cytoplasmic fragmentation (grade III >20%, grade IV >50%). These pre-embryos are only rarely used for transfer and never frozen, and will hereafter be called surplus pre-embryos.
The surplus pre-embryos, to be discarded on day 2, were instead cultured in separate microdrops of medium until the next day, when their cell number and morphology were again documented as described above. The pre-embryos were then divided into two groups: surplus I: pre-embryos having >5 blastomeres and an improved morphology from day 2; surplus II: pre-embryos with 5 blastomeres and/or that maintained their poor morphology from day 2 (Figure 1). The pre-embryos were transferred on day 3 into rS2 medium (Vitrolife Ltd, Gothenburg, Sweden) for further culture. Pre-embryos which reached the blastocyst stage on day 6, or earlier, were graded morphologically, photographed and their diameter was measured. Grade A blastocysts are expanded or expanding with a distinct trophectoderm and eccentrically located inner cell mass (ICM). Grade B blastocysts are either poorly expanded and/or with less defined trophectoderm and ICM cells but not showing any signs of degenerative foci. Grade C blastocysts exhibit poor morphology, characterized by a number of degenerative foci in the ICM and trophectoderm and a poorly developed blastocyst cavity (Figure 2). The control pre-embryo group was obtained by thawing GQE (i.e. grade I or II) frozen on day 2 using a slow-freezing method with propanediol as a cryoprotectant (Lassalle et al., 1985
). Although the optimal control group would have been fresh GQE cultured to the blastocyst stage, this was not possible due to the ethical implications involved as all GQE are either transferred or frozen at day 2 in our programme. Only pre-embryos where
75% of the cells survived the freezethaw procedure and resumed cleavage were included. Directly after the thawing procedure, control pre-embryos were cultured in IVF-20 until day 3, when the cleavage status was checked and the pre-embryos were moved to rS2 for culture to day 6. Only blastocysts meeting the criteria set out above were then included in the FISH analysis.
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FISH protocol
The slides were washed for 5 min in 1xphosphate-buffered saline (PBS) followed by an ethanol series of 70, 85 and 99.5% for 1 min each. A pepsin treatment was performed in order to increase probe penetration. Briefly, the slides were immersed in pre-warmed 0.01 N HCl containing a final concentration of 0.005% pepsin and incubated for 15 min at 37 ± 1°C followed by two rinses in water and PBS. The slides were then immersed in Carnoys fixative for 10 min at 4 ± 1°C and rinsed twice with PBS and distilled water, dehydrated in an ethanol series and air-dried. For simultaneous detection of five chromosomes, a multi-colour kit was used (MultiVysionTM PB Multi-colour Probe Panel; Vysis, Inc., Downers Grove, IL, USA) which includes chromosomes 13 (spectrum red), 16 (spectrum aqua), 18 (spectrum blue) 21 (spectrum green) and 22 (spectrum gold). A small amount (0.3 µl) of the probe was pipetted onto the nuclei, a cover glass applied and glued and the preparation denatured at 69 ± 1°C for 7 min on a heating plate. The slides were incubated at 38 ± 1°C for 4 h in a moist chamber, the cover glasses were then removed and the slides washed at 42 ± 1°C in 50% formamide + 2xstandard saline citrate (SSC) (15 min) followed by 2xSSC (10 min) and 2xSSC + 0.001% Igepeal (5 min) to remove non-specific staining. The slides were thereafter air-dried, antifade solution applied and the preparation sealed with a cover glass. The nuclei were observed with a Nikon epifluorescence microscope equipped with appropriate filters and overview pictures (x100) of the whole blastocyst were taken. These pictures together with drawn maps, allowed us to identify each nucleus and to determine the exact position of each nucleus after the second round of FISH. Before the second FISH round, the slides were washed for 5 min each in 1xPBS, 2xSSC with 0.5% Tween and 2xSSC. The slides were kept on a slow shaking device during this time. After dehydration in an ethanol series, a probe solution for chromosomes 18 (spectrum aqua), X (spectrum green) and Y (spectrum red) was applied (Vysis, Inc.). The FISH procedure was performed as described above except that the incubation time was prolonged to 18 h to increase probe attachment. After the wash, an antifade solution containing [4,6-diamidino-2-phenylindole (DAPI) II] was applied.
Signal and data analysis
The FISH-scoring criterion was that signals had to be at least one signals width apart to be scored as two separate signals. A nucleus was considered normal when all seven analysed chromosomes were present in correct numbers.
The FISH system was tested on normal lymphocytes and two human embryonic stem cell lines with normal karyotypes. FISH was performed in two rounds and the probe for chromosome 18 was included in both rounds to act as an internal control of nuclear quality and FISH efficiency. In the control series, the discordance between the first and second analysis was never >20%. Hence, this was the limit chosen for acceptance of the FISH analysis of the blastocysts.
It was found that the efficiency of the chromosome 16 probe was lowered due to repeated freezingthawing of the probe vial, although control slides showed no such effect on the other probes included in the kit. Therefore, in 13 of the blastocysts the results from chromosome 16 were excluded. A sensitivity analysis showed that this had no effect on the overall differences between or within the study groups.
Statistical methods
Distributions of the variables are given as means, SD, medians and ranges. For comparisons between groups, Fishers exact test was used for dichotomous variables and the MannWhitney U-test for ordered and continuous variables. Spearmans rank correlations test was used for all correlation analyses.
A multivariate analysis of covariance of % normal cells per blastocyst between the surplus and control group was performed, with adjustment for maternal age, aspirated oocytes, GQE and IVF/ICSI. Since the distribution of % normal cells per blastocyst was not normally distributed, it was first transformed using the inverse cumulative normal function on the empirical distribution function. All significance tests were two-tailed and conducted at the 5% significance level.
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Results |
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A multivariate analysis of covariance, based on subjects, between surplus and control group showed that the control group had significantly (P = 0.0046) higher values of % normal cells per blastocyst after adjustment for maternal age, IVF/ICSI, aspirated oocytes and number of GQE.
The detailed results from the FISH analysis of the different chromosomal aberrations on a cellular level, in relation to developmental and morphology groups, are shown in Tables V and VI respectively. From the 58 blastocysts, 6425 cells were analysed in the first FISH round and 6164 cells in the second. In general, there is an 10% overall difference observed between the surplus and the control group when the individual chromosome aberration rate is compared (Table V). By far the most common overall aberration is tetrasomy with a large difference between both developmental and morphological groups (Table VII). As seen in Table VII, 24% (14/58) of the blastocysts were mosaic with one diploid and one tetraploid (2n/4n) cell line. However, the frequency of polyploidy in each blastocyst was rather low [mean = 19.7% (SD 15.2%), range: 762%]. Overall, chromosome Y has a lower aberration rate compared with other chromosomes (Tables V and VI). Interestingly, we found one blastocyst that had extensive monosomy 22 (79/119 cells).
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Discussion |
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These results confirm that the morphological and developmental criteria used in the IVF laboratory for pre-embryo selection on day 2 are mainly correct, since this selection results predominantly in apparently chromosomally normal blastocysts (i.e. the control group). The concept of culturing surplus pre-embryos to the blastocyst stage and relying on these surplus blastocysts being chromosomally compatible with blastocysts from GQE is therefore incorrect. Several studies have shown that surplus cleavage stage pre-embryos have a high degree of aneuploidy (Munné et al., 1993, 1994; Márquez et al., 1999
) and low pregnancy and implantation rates (Puissant et al., 1987
; Steer et al., 1992
; Ziebe et al., 1997
).
Interestingly, no differences were found in mean cell number per blastocysts between the experimental groups and the total cell number was higher than reported in blastocysts derived from similar pre-embryos (Hardy et al., 1989).
It is, however, somewhat surprising that the surplus I group, which was defined as morphologically and/or developmentally improved on day 3, did not produce blastocysts of higher quality, since the same primary embryonic parameters as are used to score conventional cleavage-stage pre-embryos were applied. Perhaps morphology and/or synchronicity in developmental speed matters less in the later stages of pre-embryonic development compared to the first two or three cell divisions. In the human pre-embryo, it is known that the transition between the maternal genome and the pre-embryos own transcriptional activity occurs at this stage in development (Braude et al., 1988). It is therefore possible that a decline in developmental progress and/or morphological appearance due to suboptimal genetics might not occur until later, i.e. at the blastocyst stage, and is therefore not useful as a selection tool between day 2 and day 3. This is supported by findings that indicate that extended culture until day 3 to select pre-embryo for transfer does not increase the success rate (Laverge et al., 2001
).
In this paper we have used probes specific for chromosomes X, Y, 13, 16, 18, 21 and 22. It is, however, most probable that other chromosomes are also involved in aneuploidies in blastocysts. Which chromosomes that are most commonly represented in aneuploidies in early blastocysts is still unknown (Bahce et al., 1999; Gianaroli et al., 2001
) as chromosomal studies on blastocysts so far have been performed with a limited set of FISH probes. Recently, there have been reports where comparative genomic hybridization has been used to study pre-embryos (Voullaire et al., 2000
; Wells and Delhanty 2000
; Malmgren et al., 2002
). This technique will enable us to draw conclusions about the aneuploidy status for all chromosomes in the pre-embryo.
Our findings confirm the results reported by Sandalinas et al. where surplus pre-embryos which were already diagnosed by PGD were cultured until day 56 and where it was also found that chromosomally abnormal pre-embryos were able to develop into blastocysts but at a lower rate than normal pre-embryos. What is interesting in the present study is that 42% (19/45) of the surplus blastocysts can be considered chromosomally normal, when assessed for seven chromosomes (Table VII). This indicates that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, many surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded. However, a chromosomally normal blastocyst does not in itself always lead to implantation and development of a healthy offspring. Nevertheless, it has been shown in several studies that pre-embryonic viability is strongly correlated to chromosomal normality (Gianaroli et al., 1997; Delhanty 2001
).
In a recent study by Balaban et al. (2001), it was shown that both pregnancy and implantation rates increased when prolonging the culture of surplus pre-embryos to day 5, i.e. transferring blastocysts derived from these poor-quality cleavage stage pre-embryos. Nevertheless, their implantation rates were low as compared with other blastocyst programmes using GQE (Gardner et al., 1998a
; Jones et al., 1998
), suggesting that a higher proportion of the surplus blastocysts were chromosomally abnormal.
Many studies have shown that pre-embryos with serious chromosomal abnormalities are not compatible with blastocyst development (Benkhalifa et al., 1993; Munné et al., 1994
; Evsikov and Verlinsky, 1998
; Márquez et al., 1999
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000
; Sandalinas et al., 2001
; Bielanska et al., 2002
). But, even if the gravity of the chromosomal imbalance in blastocysts is reduced compared to cleavage stage pre-embryos, there still remains the problem of how to choose the blastocyst able to give rise to a healthy individual, especially if single embryo transfer is preferred. In the absence of a non-invasive method for selecting the chromosomally normal blastocyst, one method may be to perform aneuploidy screening (PGD-AS) at the 8-cell stage and transfer only pre-embryos/blastocysts with a normal chromosomal set-up. This method has been shown to be successful by other investigators (Gianaroli et al., 1997
; Pergament et al., 2001
).
The frequency of normal cells, regarding each separate chromosome pair in the surplus group, was higher than the results for each cell, when all the seven chromosomes are combined [7892% (Table V) versus 60% (Table IV)]. In the surplus group, the chromosomal errors are more scattered, i.e. influencing a higher number of cells, compared with the control group. In the GQE group, on the other hand, the errors are accumulated in a smaller number of cells. This may be less detrimental for the whole blastocyst, possibly enabling it to discard a few chromosomally abnormal cells without loosing developmental capacity. These results are in concordance with our previous findings where we found that a certain morphological aberration (uneven blastomeres) resulted in a higher incidence and a more widespread distribution of the chromosomal aberrations (Hardarson et al., 2001).VI
The finding that 24% of the blastocysts had two cell lines with two different ploidy stages 2n/4n (Table VII) may not be surprising as it has been suggested that tetraploidization is a part of normal trophoblast development into syncytiotrophoblasts (Drury et al., 1998; Plachot, 1998
; Bielanska et al., 2002
). Furthermore, as this 2n/4n mosaicism may appear normally as a low degree mosaicism (Clouston et al., 1997
) some of these blastocysts may have been regarded as normal due to the 10% FISH error threshold (see Table VII).
We found that good blastocyst morphology is correlated both to higher cell number per blastocyst and to a higher percentage of chromosomally normal cells, based on the seven chromosomes analysed. Choosing a blastocyst of good morphology would therefore most likely increase the chance of implanting. The degree of blastocyst development in the surplus groups in this study was comparable to that in other studies using surplus pre-embryos (Shoukir et al., 1998; Balaban et al., 2001
). In total, we found no differences in blastocyst formation rate between pre-embryos from conventional IVF compared with ICSI pre-embryos, although there were differences within the groups (Table II). Our control group had a numerically higher blastocyst rate than the surplus groups (31.4 versus 22.6%) but this difference was not significant (P = 0.14). This rate is still low compared with other studies, where between 45% (Marek et al., 1999
) and 66% (Gardner et al., 1998a
) blastocyst developmental rates have been reported using sequential media, as in the present study. One explanation for this difference may be that we used frozenthawed pre-embryos, which are known to have an impaired developmental capacity and high aneuploidy rates (Iwarsson et al., 1999
; Edgar et al., 2002
). However, this effect might be partially offset by the fact that many of the patients who donated their frozen pre-embryos had delivered a healthy child from the same pre-embryo cohort, indicating a high pre-embryo capacity. We believe that we are at least not underestimating the difference between an optimal control group (i.e. fresh good quality pre-embryos) and a surplus group.
In conclusion, we found in this study that blastocysts from morphologically surplus pre-embryos are on average more chromosomally and morphologically abnormal than blastocysts developing from GQE, when assessed for seven chromosomes. Nevertheless, according to the present study, 42% of these surplus blastocysts are chromosomally normal, based on the seven chromosomes analysed, and further studies are needed to find factors that can help to distinguish between the abnormal and normal ones. The present clinical practice in our IVF laboratory is to discard surplus pre-embryos on day 2 or 3. By prolonged culture combined with PGD-AS, a large number of these pre-embryos may be proved normal, with a theoretically high implantation rate. The benefits of such methods must be weighed against the extra costs associated with prolonged pre-embryo culture and PGD-AS and should perhaps only be used for selected patient groups. The possibility of increasing the number of pre-embryos available for IVF patients by prolonged culture surplus of pre-embryos should be taken into due consideration, also in light of the single embryo transfer discussion.
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Acknowledgements |
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Submitted on July 1, 2002; resubmitted on October 24, 2002; accepted on November 11, 2002.