Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial

Catherine Staessen1,3, Peter Platteau1, Elvire Van Assche2, An Michiels2, Herman Tournaye1, Michel Camus1, Paul Devroey1, Inge Liebaers2 and André Van Steirteghem1

1 Centre for Reproductive Medicine and 2 Centre for Medical Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium

3 To whom correspondence should be addressed. Email: catherine.staessen{at}az.vub.ac.be


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It is generally accepted that the age-related increased aneuploidy rate is correlated with reduced implantation and a higher abortion rate. Therefore, advanced maternal age (AMA) couples are a good target group to assess the possible benefit of preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) on the outcome after assisted reproductive technology (ART). METHODS: A prospective randomized controlled clinical trial (RCT) was carried out comparing the outcome after blastocyst transfer combined with PGD-AS using fluorescence in situ hybridization (FISH) for the chromosomes X, Y, 13, 16, 18, 21 and 22 in AMA couples (aged ≥37 years) with a control group without PGD-AS. From the 400 (200 for PGD-AS and 200 controls) couples that were allocated to the trial, an oocyte pick-up was performed effectively in 289 cycles (148 PGD-AS cycles and 141 control cycles). RESULTS: Positive serum HCG rates per transfer and per cycle were the same for PGD-AS and controls: 35.8% (19.6%) [%/per embryo transfer (per cycle)] and 32.2% (27.7%), respectively (NS). Significantly fewer embryos were transferred in the PGD-AS group than in the control group (P<0.001). The implantation rate (with fetal heart beat) was 17.1% in the PGD-AS group versus 11.5% in the control group (not significant; P=0.09). We observed a normal diploid status in 36.8% of the embryos. CONCLUSIONS: This RCT provides no arguments in favour of PGD-AS for improving clinical outcome per initiated cycle in patients with AMA when there are no restrictions in the number of embryos to be transferred.

Key words: age/aneuploidy screening/FSH/preimplantation genetic diagnosis/RCT


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
For an infertile couple, the primary aim of assisted reproductive technology (ART) is to obtain the birth of one healthy child. During these last few years, concerted efforts focused on reducing multiple pregnancy after ART by restricting the number of embryos to transfer. Therefore, one of the crucial steps in an ART programme is the selection of the embryo with the highest implantation potential. Different strategies can be considered for selecting embryos with the highest implantation potential for transfer.

One strategy involves microscopic evaluation of morphological criteria associated with improved viability such as fragmentation, cell number, cell size, multinucleation of the blastomeres and early cleavage (Cummins et al., 1986Go; Claman et al., 1987Go; Puissant et al., 1987Go; Staessen et al., 1992Go; Steer et al., 1992Go; Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Hardarson et al., 2001Go; Van Royen et al., 2001Go; Salumets et al., 2003Go) or by assessing pronuclei disposition and nucleolar organization (Scott et al., 2000Go; Nagy et al., 2003Go). Another strategy involves culture to the blastocyst stage, thereby allowing self-selection of those embryos capable of proceeding to blastulation and exclusion of less viable embryos showing developmental arrest (Gardner et al., 1998Go). Genetic analysis has shown that many human preimplantation embryos show chromosomal abnormalities which may affect the outcome of ART (Munné et al., 1994Go, 1995Go, 1998bGo; Harper et al., 1995Go; Laverge et al., 1997Go; Magli et al., 2001Go). Single-cell multicolour fluorescence in situ hybridization (FISH) has been developed, allowing interphase chromosomes to be analysed rapidly. The possibility of detecting various chromosomes simultaneously in a single cell resulted in the development of preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Using PGD-AS, it has become possible to analyse the ploidy status of several chromosomes from in vitro embryos before transfer.

Besides the morphological quality and the dynamics of the cleavage pattern up to the blastocyst stage of the cultured human embryo in vitro, PGD-AS has been introduced as another approach by which to define the right embryo for transfer. The hypothesis is that exclusion of aneuploid embryos for transfer might improve implantation, reduce abortion rates and avoid the birth of children with numerical chromosomal abnormalities after ART, and this has been suggested in several comparative studies (Gianaroli et al., 1999Go; Munné et al., 1999Go, 2003Go).

Increasing aneuploidy rates in oocytes and embryos in relation to increasing maternal age have been demonstrated (Munné et al., 1995Go; Dailey et al., 1996Go; Marquez et al., 2000Go). It is well known that the age-related increase in aneuploidy rate is correlated with a reduced implantation and a higher abortion rate. Advanced maternal age (AMA) patients, here defined as ≥37 years, are a good target group to assess the possible benefit of aneuploidy screening. The aim was to conduct a randomized controlled trial (RCT) to investigate the effect on implantation after transferring in vitro cultured blastocyst with or without PGD for aneuploidy screening in AMA couples. Furthermore, the non-transferred or non-cryopreserved embryos were analysed further to investigate the accuracy of the diagnosis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study design
From March 2000 to December 2003, a prospective RCT was performed on AMA couples. This RCT was set up to compare the outcome in AMA couples (aged ≥37 years) after blastocyst transfer including PGD-AS, using FISH for the chromosomes X, Y, 13, 16, 18, 21 and 22, with a control group after blastocyst transfer without PGD-AS. In order to double the anticipated implantation rate in this AMA group of 15% per embryo, we calculated that at least 157 embryos had to be transferred in each group ({alpha} 5%, {beta} 10%).

During consultation, couples fulfilling the following inclusion criteria were asked by the gynaecologist to participate in this RCT: maternal age of 37 years or older, need for ICSI with motile sperm and both partners with a normal karyotype. Once they had given written informed consent, eligible couples were assigned randomly for ICSI followed by blastocyst transfer with or without PGD-AS. As for PGD, the consent form mentioned that patients agreed to donate embryos for research that were chromosomally abnormal or unsuitable for transfer or cryopreservation. The study was approved by the Ethics Committee of the University Hospital.

A total of 400 (200 for PGD-AS and 200 controls) couples were recruited to participate in the trial (Figure 1). Eventually, in 289 couples (141 control cycles and 148 PGD-AS cycles), an oocyte pick-up was performed effectively. The reasons for cancelling the intended treatment cycle were similar in both the control group and the PGD-AS group.



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Figure 1. Patient flow through the randomized controlled trial.

 
Ovarian stimulation, oocyte retrieval and embryo culture
All female partners were superovulated using a GnRH analogue suppression protocol (Kolibianakis et al., 2002Go) in combination with HMG. The ovulation was induced by the administration of 10 000 IU of HCG.

Transvaginal ultrasound-guided oocyte retrieval was scheduled 36 h after HCG administration. ICSI was used to fertilize the oocytes. The ICSI procedure on mature metaphase II oocytes was performed as described by Joris et al. (1998)Go. Microinjected oocytes were washed and incubated in 25 µl droplets of medium in a Petri dish at 37°C in an incubator containing 5% CO2, 5% O2 and 90% N2. Sequential media from Vitrolife, Göteborg, Sweden (G-Fert, G2.1 and G2.2) were used throughout the study.

Assessment of fertilization and embryo evaluation
Fertilization was assessed 16–18 h after injection (Staessen et al., 1995Go). Normal fertilization was confirmed by the presence of two distinct pronuclei and polar bodies at x400 magnification using an inverted microscope

Embryo evaluation was performed on days 2 and 3 by recording the number of blastomeres and percentage of fragmentation. Grade A embryos were defined as embryos without anucleate fragments. Grade B embryos had blastomeres of equal or unequal sizes, with a maximum of 20% of the volume of the embryo filled with anucleate fragments. In grade C embryos, anucleate fragments were present in 20–50% of the volume of the embryo. The embryos were evaluated daily until day 5. On day 5, the embryos were classified into arrested embryos showing no signs of compaction on day 5, compacting embryos (C1–C2), early blastocysts (Bl1–Bl2) and expanding blastocysts (Bl3–Bl7) according to the classification proposed by Gardner and Schoolcraft (1999)Go.

Embryo biopsy
Embryos of grade A, B or C with at least five blastomeres were biopsied (Joris et al., 2003Go) in the morning of day 3 after injection. The selection criteria that were used to decide whether an embryo was suitable for biopsy were similar to those used to decide whether an embryo was transferable on day 3 in the regular ICSI programme without PGD. Before biopsy, the blastomeres were checked for the presence of nuclei. From the 6-cell stage onwards, two blastomeres per embryo were removed (Van de Velde et al., 2000Go); otherwise, one blastomere was removed.

Embryo biopsy was carried out in HEPES-buffered medium under oil. The embryos were first incubated in calcium–magnesium-free medium EB10 (Scandinavian IVF Science, Göteborg, Sweden) before biopsy. Laser technology (Fertilase®) was used to drill a hole of ~30 µm in the zona pellucida (on average of 2–3 pulses of 7 ms were applied), and one or mostly two nucleated blastomeres were gently aspirated with an aspiration pipette (±40 µm outer diameter).

Spreading of the interphase nuclei and FISH procedure
Using a mouth pipette, the individual blastomeres were first rinsed in medium and then transferred to a 1 µl droplet of 0.01 mol/l HCl/0.1% Tween-20 solution (Coonen et al., 1994Go; Staessen et al., 1996Go) on a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany). The nuclear content of both blastomeres from the same embryo was fixed on the same slide in very close proximity.

A two-round FISH procedure was carried out as described previously (Staessen et al., 2003Go), allowing us to detect chromosomes X, Y, 13, 18 and 21 (round 1), and 16 and 22 (round 2). In the first round, probes for chromosomes X, Y, 13, 18 and 21 (DXZ1, Spectrum Blue; DYZ3, SpectrumGold; LSI13, SpectrumRed; D18Z1, SpectrumAqua; LSI21 SpectrumGreen; Multivision PGT Probe Panel; Vysis Inc. Downers Grove, IL) were used. The second hybridization solution was prepared by mixing a probe for chromosome 16 (Vysis, Satellite II DNA/D16Z3 probe, Spectrum Orange) and a probe for chromosome 22 (Vysis, LSI 22, 22q11.2, Spectrum Green). The FISH images were captured using a computerized system. All the slides were observed and interpreted again by two independent observers.

FISH scoring criteria
The specific FISH signals detected in a given blastomere were interpreted as follows: (i) a blastomere was considered to be diploid normal when two gonosomes and two chromosome 13-, 16-, 18-, 21- and 22-specific signals were present; (ii) a blastomere was considered to be haploid, triploid or tetraploid when one, three or four signals, respectively, for the investigated chromosomes were present; (iii) a blastomere was considered to be aneuploid when an extra (trisomic) or missing (monosomic) signal for one chromosome was observed, in the presence of two signals for the remaining chromosomes analysed; (iv) a blastomere was considered as double aneuploid when for two different chromosomes an extra or missing signal was observed in the presence of two signals for the remaining chromosomes; and (v) a blastomere was considered as combined abnormal when it was neither diploid nor haploid, triploid, tetraploid or aneuploid.

After FISH analysis, only embryos found to be chromosomally normal after analysis of one or two blastomeres were transferred on day 5.

Embryo transfer
In the PGD-AS group, the transfer was performed on day 5 if at least one compacting embryo or early blastocyst was obtained from the embryos found to be genetically normal. In the control group, the transfer was also performed if at least one compacting embryo or early blastocyst was obtained on day 5; six transfers were wrongly planned on day 3. In line with the transfer policy at our centre, up to three blastocysts were transferred when the patients were between 37 and 39 years old; and, in patients of ≥40 years of age, up to a maximum of six blastocysts were transferred (Adonakis et al., 1997Go; Grimbizis et al., 1998Go).

Pregnancy
Two increasing HCG concentrations >10 IU/l at least 10 days following embryo transfer were considered positive. A clinical pregnancy was defined by the presence of an intra-uterine gestational sac with positive fetal heart activity at ultrasonography performed at least 6 weeks post-embryo transfer. As ongoing implantation, the number of implantations developing beyond 12 weeks was counted.

End-point
The primary outcome was embryo implantation rate. The implantation rate represents the ratio between the number of gestational sacs with a fetal heartbeat and the total number of embryos transferred.

All blastomeres of the embryos donated for research were analysed further to confirm the diagnosis and to determine the accuracy of the PGD-AS analysis. When the abnormality detected at diagnosis cannot be found in any cell after re-analysis, we consider it as non-confirmed.

Statistical analysis
Data were analysed by the Student's t-test and {chi}2 analysis and considered significant if P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical outcome
The control group included 141 couples, while the study group included 148 couples. As shown in Table I, the mean female age in the PGD-AS group was 40.1±2.4 years versus 39.9±2.4 years in the control group (NS). There was no significant difference between patients in the control and PGD-AS groups with regard to years of infertility, G/P status where G status = number of gestations and P status = number of parity (i.e. number of pregnancies of >20 weeks gestation), trial rank, aetiology of infertility or treatment cycle embryology end-points such as number of eggs retrieved, fertilization rate or rate of progression to ≥6-cell type 1–3.1 on day 3.


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Table I. Characteristics and treatment cycle-related embryology

 
The remaining clinical results are summarized in Table II. It is obvious that the number of cycles with embryo transfer was decreased in the PGD-AS group (54.7%) as compared with the control group (85.8%) (P<0.001). Transfer was cancelled in 67 cycles of the PGD-AS group. The reasons for that are shown in Table II.


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Table II. Clinical results

 
In the PGD-AS group, 164 embryos diagnosed to be normal by FISH and which had cleaved further were transferred, i.e. a mean of 2.0±0.9 embryos per transfer. In the control group, a total of 338 embryos were transferred, i.e. a mean of 2.8±1.2 embryos per transfer (P<0.001). In the control group in patients above 40 years, nine transfers with five or six embryos were performed from which in three cases a pregnancy was obtained: one preclinical abortion, one ongoing singleton and one ongoing twin pregnancy. In the tested group, only one patient received five embryos resulting in a preclinical pregnancy.

Between the control group and the PGD-AS group, the distribution of the quality of the transferred embryos was similar: in the control group, 35.9% of the transferred embryos were at the compacting stage (C1–C2) versus 26.0% in the PGD-AS group, 34.4% in the control group were Bl1–Bl2 versus 37.0% in the PGD-AS group, and 29.7% of the embryos in the control group were Bl3–Bl7 versus 37.0% in the PGD-AS group (NS). Positive serum HCG rates per transfer and per cycle were similar in both groups [% positive serum HCG/per embryo transfer (per cycle)]: controls 32.2% (27.7%) and PGD-AS 35.8% (19.6%) (NS). The odds ratio was 0.71 [95% confidence interval (CI) 0.41–1.23]. In the control group as well as in the PGD-AS group, similar rates of abortions were observed; respectively 25.6% (10 out of 39) and 24.1% (seven out of 29) ended in pregnancies not developing beyond 12 weeks. The implantation rate (gestational sac + fetal heart activity) was 17.1% in the PGD-AS group versus 11.5% in the control group (not significant; P=0.09). The odds ratio was 1.48 (95% CI 0.87–2.5) with a number needed to treat of 18.1 (95% CI 8.2–{infty}). The percentage of ongoing implantations beyond 12 weeks was 10.4% in the control group and 16.5% in the PGD-AS group (P=0.06; NS). At the time of writing, the control group has yielded 23 singleton and five twin pregnancies already delivered, and one twin ongoing pregnancy. The PGD-AS group has produced 16 singleton, two twin and one triplet pregnancy already delivered and two ongoing singleton pregnancies. One twin pregnancy expulsed at 21 weeks. All the children born were healthy.

Genetic analysis
The FISH results obtained after PGD-AS diagnosis as well as the results for the re-analysed embryos are summarized in Table III. Of 685 embryos suitable for biopsy, we obtained a FISH result in 653 (95.3%) embryos. In 112 (17.2%) embryos, the diagnosis was based on the analysis of one blastomere and in 541 (82.8%) it was based on the analysis of two cells. In 240 embryos (36.8%) we observed a normal diploid status, in 353 embryos (54.1%) different abnormalities were observed and in 60 (9.2%) embryos we found a normal nucleus in one blastomere and an abnormal nucleus in the other blastomere. From the normal embryos (n=240), 164 were transferred and nine were frozen. The remaining 67 embryos were developmentally or morphologically not suitable for transfer and/or cryopreservation and were re-analysed; the FISH re-analysis was successful in 43 embryos and all could be confirmed as being normal in the majority of the cells (0% false negative). A total of 285 embryos out of the 413 diagnosed as abnormal were re-analysed successfully (Tables III and IV); in 261 embryos (91.6%) the initial diagnosis could be found in at least a proportion of the cells, while in 24 embryos the abnormal cells could not be found and the embryo was grossly normal (8.4% false positive). From the 261 genetically abnormal embryos with confirmed re-analysis, we estimate that in 153 (58.6%) it concerns meiotic errors (uniform trisomies and monosomies), in 101 (38.7%) it concerns post-zygotic mitotic errors (mosaics and combined abnormalities) and in seven (2.7%) it concerns fertilization errors (haploid, triploid and tetraploid). The largest proportion of non-confirmed diagnoses were the monosomies and the mosaics including a monosomy (19 out of 24: 79.2%). Another observation was that in 17.5% (50 embryos) of the embryos diagnosed as abnormal, besides the detected abnormal cells, there were also normal cells indicating that these embryos were mosaic with a normal cell line in combination with one or more abnormal cell lines. In the PGD-AS group, a total of 49 patients did not receive a transfer. From the 38 cycles where all the embryos were genetically abnormal, in two cycles after re-analysis we detected in fact genetically normal embryos. Also, from the 11 patients where there was no morula or blastocyst formation from genetically normal embryos, in one case after re-analysis of the genetically abnormal embryos we found one normal embryo. These give us in total three out of 49 (6%) cycles where in fact a transfer was not done due to a false-positive result.


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Table III. FISH results at diagnosis and after analysis of the embryos donated for research

 

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Table IV. Re-analysis of genetically abnormal embryos: incidence of embryos with abnormal cells in combination with normal cells

 
Figure 2 represents the incidence of normal and abnormal embryos in relation to the cell stage on day 3 (Figure 2A) and the developmental stage on day 5 (Figure 2B). On day 3, of the total number of 653 embryos from which we obtained a diagnosis, 61 had <6 cells, 92 were at the 6-cell stage, 101 were at the 7-cell stage, 283 were at the 8-cell stage and 116 had >8 cells. The overall distribution of the abnormal embryos was not significantly different with relation to the cell stage on day 3 although, if we compared the results for each cell stage separately, the 8-cell stage has a significantly higher incidence of being normal (P<0.01). In 645 embryos from which the development was documented until day 5, a significant increase of normal embryos was observed in relation to the further developmental stage on day 5 (P<0.01). The results indicate that if we can select an expanded blastocyst on day 5, 65% of them are genetically normal versus 41.3% if we select an 8-cell stage embryo on day 3. If no expanding blastocysts are obtained and we have to select among the compacting and non-expanding blastocysts, there is no difference from the selection on day 3. Comparing the different types of abnormalities, there is no tendency towards differences in the different type of abnormalities observed on day 3, whereas in day 5 embryos trisomies and mosaics are seen to develop until the expanding blastocyst stage, while combined abnormalities do not develop further in vitro. Statistical analysis to compare each type of anomaly could not be performed because of the low numbers in each category.



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Figure 2. Relationship between the FISH diagnois and the developmental stage on day 3 (A) and on day 5 (B).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Several ART centres have introduced PGD-AS to increase their clinical success rates. So far, however, only one controlled study including only 19 AMA patients has been published (Werlin et al., 2003Go) together with three additional comparative studies (Gianaroli et al., 1999Go; Munné et al., 1999Go, 2003Go). Here we present the first large-scale prospective RCT that investigates the effect of the association of in vitro blastocyst culture and PGD-AS for the selection of the best embryos in AMA couples, i.e. ≥37 years of age. A total of 400 patients were recruited. In 289 cycles, an oocyte pick-up was performed effectively. The main reasons for drop-out were no adequate ovarian response and the decision taken by the couple to abandon further fertility treatment

At the clinical level, no differences in outcome were observed between the PGS-AS group and the control group either per cycle or per transfer, which indicates that PGD-AS does not improve the pregnancy rate. These results are in agreement with previous studies, because none of the earlier reports demonstrated an improvement of the clinical pregnancy rate (Gianaroli et al., 1999Go; Munné et al., 1999Go, 2003Go). In the PGD-AS group, in which in 25.7% of the cycles all embryos were found to be abnormal, a significantly higher number of cycles did not have a transfer. Also in the small series reported by Werlin et al. (2003)Go, 28.6% of patients had no normal embryos to transfer. This verdict, however, provides valuable diagnostic information for couples to redirect their future fertility treatment towards other alternatives such as oocyte or embryo donation and/or adoption. For patients with a transfer, the same pregnancy rate per transfer was observed in the PGD-AS group despite the fact that significantly fewer embryos were transferred, indicating that suitable embryos were selected for transfer.

In this study, the implantation rates after PGD-AS (17.1%) and in the control group (11.5%) were similar (NS). The number that needed to be treated indicates that 18 genetically normal PGD-AS embryos have to be transferred to obtain one extra viable fetus. Other studies have reported inconsistent results concerning the impact of PGD-AS on implantation rates in AMA couples. In two studies, a significant improvement in implantation rate was observed after PGD-AS in AMA patients compared with a matched control group. In the report by Gianaroli et al. (1999)Go, aneuploidy screening for 6–8 chromosomes was performed in 73 AMA (≥36 years of age) patients, and compared with 84 controls who underwent assisted zona hatching. The implantation rates were 25.8% in the tested group and 14.3% in the control group (P<0.01). In a recent paper, Munné et al. (2003)Go compared 138 cycles with PGD-AS for nine chromosomes (X, Y, 13, 15, 16, 18, 21, 22 and 1 or 7, or 14 or 17) with a matched control group and found that AMA (≥35 years of age) showed a higher implantation rate (18%) than their matched controls (11%) (P<0.05%). However, in a previous publication, Munné et al. (1999)Go observed no significant difference in implantation rates after aneuploidy testing for 6–8 chromosome pairs in patients aged >35 years undergoing IVF in a multi-centre comparative study (17.6% in the test group versus 13.7% in the control group). In the three comparative studies (Gianaroli et al., 1999Go; Munné et al., 1999Go, 2003Go), the transfers were done on either day 3 or 4. It is very difficult to compare results reported in the literature because of differences in patient selection and methodology that may influence outcome.

In our study, fewer zygotes were obtained in our patients than in the study by Munné et al. (2003)Go. Munné mentioned that no difference in implantation was observed when patients had less than eight zygotes available. Another major difference between this RCT and the comparative studies are that two blastomeres are mostly removed, whereas in most comparative studies only one blastomere is biopsied. However, it has been demonstrated that neither the blastocyst formation rate (Lamb et al., 2002Go) nor the implantation rate (Parriego et al., 2003Go) are significantly different when one or two cells have been biopsied. Since laboratory intrinsic factors can interfere, an RCT comparing the results after removal of one cell or two cells is currently being conducted at our centre.

In different studies, the prevalence of chromosomal abnormalities in embryos from AMA patients is found to vary from 40.4 to 64.0%, depending on the number of chromosomes being investigated (Munné et al., 1998aGo; Gianaroli et al., 1999Go; Pellicer et al., 1999Go; Kahraman et al., 2000Go). This study confirms that AMA patients have a high proportion of genetically abnormal embryos despite the fact that these embryos are morphologically normal on day 3. Another difference between this study and other reports is that we transferred compacting and blastocyst-stage embryos on day 5, introducing an additional criterion for embryo selection. The effect of the genetic constitution on further embryonic development has been analysed (Magli et al., 2000Go; Sandalinas et al., 2001Go) and it was reported that aneuploid embryos have a lower capacity to develop to morulae and blastocysts than euploid embryos, although an aberrant genome is not incompatible with development to the blastocyst stage (Clouston et al., 1997Go; Evsikov and Verlinsky, 1998Go; Sandalinas et al., 2001Go). We also observed that in patients where expanded blastocysts are obtained, the chance of selecting a chromosomally normal embryo increased from 41.3% on day 3 to 65% on day 5. The high pregnancy rate in the control group may thus be explained by selection towards more genetically normal embryos reaching the blastocyst stage. This study also confirms that, as was previously demonstrated, transfer of day 5 embryos could not ensure the absence of chromosomal abnormalities (Magli et al., 2000Go; Sandalinas et al., 2001Go). By performing PGD-AS, it is possible in the single embryo transfers as well as in a number of double and triplet embryo transfers to define on the basis of the gender of the live born child which of the embryos generated the conceptus. We could demonstrate the relationship between morphological quality and implantation potential of day 5 embryos on which we also performed PGD-AS. However, in some cases, we observed that it was not the morphologically best one that implanted (Staessen et al., 2004Go). In this and other studies (Gianaroli et al., 1999Go; Munné et al., 2003Go), significantly more embryos were transferred in the control group than in the PGD-AS group. The soundness of comparing implantation rates is therefore questionable, since the denominator is different across the groups compared. In order to make a correct comparison between the control and PGD-AS group, similar numbers of embryos should be transferred in both groups. Since July 2003, there are restrictions on the number of embryos transferred depending on the age of the patient and the rank of the trial. In patients younger than 36 years, a single embryo transfer has to be done in the first two cycles; in patients older than 36 and younger than 40 years, a maximum of two embryos can be replaced in the first two cycles. Therefore, there are no ethical objections to such a study being carried out.

Another aim of PGD-AS is to reduce the abortion rate. Munné et al. (1999)Go observed that the abortion rate decreased significantly after PGD-AS (15.0 versus 33.8%; P<0.05), resulting in a significantly higher ongoing pregnancy rate (15.9 versus 10.6%; P<0.05). However, we did not observe any difference in the ongoing implantation rate between the PGD-AS and the control groups.

No chromosomal abnormalities could be detected in the children born in this study. Although in the control group a number of abnormal embryos were replaced, there was no higher incidence of miscarriages. This confirms that in the human there is a natural selection that prevents the implantation of abnormal embryos. In this study, the effect of PGD-AS on the outcome also suggests that the impact of chromosomal abnormalities on the reduced implantation with AMA is less pronounced, maybe because not all chromosomes are under investigation and because other chromosomes may be involved with implantation. Other factors such as cytoplasmic factors (Barritt et al., 2001Go), alterations of endometrial receptivity or excessive ovarian stimulation (Simon et al., 1998Go) may also play an important role in the implantation process.

It is well known that rates of chromosomal mosaicism in cleavage-stage embryos are high (Munné et al., 1994Go; Harper et al., 1995Go; Laverge et al., 1997Go; Staessen et al., 1999Go). Therefore, the biopsied cells may not be representative for the whole embryo (Los et al., 2004Go). In this study, we observed an incidence of discordance between both cells implying a normal and an abnormal nucleus in 9.3% of all the embryos investigated, or 11.1% if we consider only those where two blastomeres were analysed. In 17.5% of the re-analysed abnormal embryos, we found a normal cell line in combination with abnormal cells. Such mosaicisms can lead to misdiagnosis. A misdiagnosis of trisomy 21 has already been reported (Munné et al., 1998aGo), which may have been due to disomy/trisomy mosaicism in the embryo or to technical factors, e.g. overlapping signals, loss of nuclear material during fixation or failure of hybridization. Errors may be reduced providing a diagnosis is performed systematically on the basis of two cells and providing only embryos of which both cells are normal are transferred (Delhanty and Handyside, 1995Go). However, errors could still occur because in some embryos there may be a mixture of euploid and aneuploid cells (Voullaire et al., 2000Go), and it may still be possible to biopsy two euploid cells from an embryo that is predominantly aneuploid (Wilton, 2002Go). On the other hand, the consequences of chromosomal mosaicism for human embryonic development are unknown. Therefore, detecting and discarding mosaic embryos may imply an important loss of potentially viable normal embryos. Bielanska et al. (2002)Go suggested that the developmental potential of mosaic embryos is dependent on the type and proportion of non-diploid cells. These abnormal blastomeres will probably not survive as they are hardly ever observed in aborted material. Mosaicism involving multiple chromosomal anomalies, or a non-diploid karyotype in >50% of blastomeres per embryo, appears to reduce the capacity to develop into a blastocyst. In contrast, small numbers of aneuploid cells in early embryos may not be detrimental to successive cleavage divisions. Ziebe et al. (2003)Go analysed all blastomeres from embryos obtained from oocytes donated for research by seven-probe FISH, and found that only 31% were uniformly normal. They concluded that it is still not known to what extent chromosomal abnormalities compromise the developmental potential of the pre-embryo and whether any corrective mechanism exists within the embryo that may compensate for various degrees of chromosomal errors. So the matter of how ‘normal’ an embryo needs to be in order to implant and give rise to a healthy baby is still unresolved. Coonen et al. (2004)Go demonstrated that anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage. This may explain the high incidence of mosaics normal/monosomic and monosomies obtained in the embryos. Monosomies are rarely observed in abortions, except for the X0, and are also less frequent in blastocysts (Sandalinas et al., 2001Go). This may indicate that monosomic cells are not surviving and it may also explain why these are less frequently confirmed, although a FISH failure leading to the absence of a signal could have occurred.

Patients having a transfer after PGD-AS have an advantage since by transferring fewer embryos they obtain a similar pregnancy rate. In the case of no transfer, patients are confronted with the rejection of embryos that might have had a chance of implanting. There is no transfer for a significant number of patients because they lack normal embryos. We observed that in ~25% of the embryos rejected for transfer, there were normal cells. Although it may sound unethical, we might propose to transfer some categories of abnormal embryos after discussion with patients, since there is no clear information about the extent to which an embryo should be normal before implantation is allowed. Examples include monosomic embryos, especially for the autosomal chromosomes, the mosaics normal/autosomal monosomy and the mosaic normal/combined abnormalities, especially when they reach the blastocyst stage. Of course, all the cases involving a trisomic cell line should not be considered for transfer. Transferring such embryos would be the only way of understanding the implantation potential of such embryos.

The identification of abnormalities in a cohort of morphologically good quality embryos prevents the transfer of those embryos that are destined either not to implant or to abort spontaneously. The patients' chances may be reduced in this way. Moreover, since by transferring genetically abnormal embryos, genetically normal ones will be frozen, and since only some 50% survive thawing, the patients' chances are reduced yet again.

In conclusion, we did not find arguments in favour of PGD-AS for improving the clinical outcome per initiated cycle in AMA patients over 37 years of age when no restrictions are put on the number of embryos transferred. Our results indicate that, although we transferred a significantly lower number of embryos in the PGD-AS group compared with the control group, no differences in pregnancy rates were observed. PGD-AS, however, may be advantageous when there is a strict regulation on the number of embryos to transfer. In countries where the number of embryos for transfer is limited or restricted especially to avoid multiple pregnancies, PGD-AS can indeed be a tool to select the best embryos for transfer. Otherwise, persistent high numbers of chromosomally abnormal embryos after PGD-AS may convince couples to consider alternative options such as the use of oocyte or embryo donation and/or adoption.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank the clinical, scientific, nursing and technical staff of the Centre for Reproductive Medicine and especially the colleagues of the microinjection and IVF laboratory. We are very grateful to Marleen Carlé, Sylvie Mertens and Griet Meersdom for providing technical assis tance with FISH, and Ronny Janssens and Hubert Joris who performed the biopsies. We also wish to thank Michael Whitburn of the University Language Centre for proofreading the English text. Finally, we wish to thank Professor John Collins and Dr E.Kolibianakis for their help with the statistical analysis. This study was supported by Grants from the Belgian Fund for Scientific Research Flanders (FWO-Vlaanderen).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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