Hum. Reprod. Advance Access published online on December 22, 2007
Human Reproduction, doi:10.1093/humrep/dem327
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Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis
1 Department of Embryology and Genetics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 2 Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 3 Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 4 Veeda Clinical Research, Koning Albertlaan 160, 1082 Brussels, Belgium
5 Correspondence address. Tel: +32-2-477-60-73; Fax: +32-2-477-68-60; E-mail: karen.sermon{at}uzbrussel.be
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Abstract |
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BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand.
METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo).
RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): –3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found.
CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.
Key words: preimplantation genetic diagnosis/preimplantation genetic screening/embryo biopsy/embryo development/diagnostic efficiency
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Introduction |
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Preimplantation genetic diagnosis (PGD) can be considered as an alternative to prenatal diagnosis which circumvents the problem of therapeutic abortion for a genetic disease. It involves the genetic testing of blastomeres from preimplantation embryos followed by the selective transfer of embryos shown to be unaffected for the disease under study. The technique has become possible after the simultaneous development of IVF, micromanipulation of the embryo, the PCR for monogenic disorders (including some X-linked diseases) (Handyside et al., 1992
; Handyside, 1998; Braude et al., 2002; Sermon et al., 2004) and fluorescence in situ hybridization (FISH) for chromosomal abnormalities (Van Assche et al., 1999; Munné et al., 2000), for sexing (Staessen et al., 1999) or for aneuploidy screening (preimplantation genetic screening; PGS) (Wilton, 2002; Munné et al., 2003a; Staessen et al., 2004). PGS can be offered to couples with a low a priori risk for a child with a genetic defect who are undergoing artificial reproductive technology with the aim to improve the delivery rate with live birth. PGS is offered in cases of advanced maternal age, recurrent abortions, recurrent IVF failure and non-obstructive azoospermia (Munné et al., 2003a; Staessen et al., 2004; Gianaroli et al., 2005; Platteau et al., 2005).
The final goal of PGD is the birth of one healthy child and since genetic analysis is performed on one or two single blastomeres, it has to meet high standards of efficiency and accuracy, including allele drop-out (ADO) and contamination control (Lissens and Sermon, 1997) for PCR and an optimal FISH technique (Velilla et al., 2002; Preimplantation Genetic Diagnosis International Society, 2004; Thornhill et al., 2005).
Until 1998, at our centre, one cell was biopsied from embryos with five or six cells and two cells were biopsied if seven or more blastomeres were present. Data from the literature and from our own centre indicated that the removal of two cells from embryos containing at least seven cells had no adverse effects on the preimplantation development in the human (Hardy et al., 1990; Van de Velde et al., 2000). As with most diagnostic procedures, PGD can involve errors as reported by the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium (Sermon et al., 2007). A total of seven misdiagnoses were reported after PGD using FISH and 10 after PGD using PCR. In 1998, a misdiagnosis was detected at prenatal diagnosis at our own centre after PGD for myotonic dystrophy and the transfer of an embryo for which the genetic diagnosis had been based on only one blastomere. Since then, the genetic diagnosis of an embryo was only accepted when the same result was obtained in two biopsied blastomeres. Theoretical statistical analysis showed that the accuracy of diagnosis using two markers on one cell was comparable to the accuracy of the diagnosis of one marker on two cells (Lewis et al., 2001). Today, many simplex PCRs (amplification of one specific genomic locus) have been replaced and the majority of the newly developed tests involve multiplex PCRs in which the simultaneous analysis of two or more genetic loci increases the diagnostic accuracy significantly. For the FISH cycles, the number of chromosomes analysed increased with the development of different fluorochromes.
Ongoing discussions concerning the safety and possible detrimental effect on the embryos of a one-cell versus two-cell biopsy on the one hand and technical improvements concerning diagnostic technologies on the other, raised the following questions: first, whether the embryo development and the delivery rate with live birth would significantly differ after the removal of only one cell as compared to two cells and secondly, whether we can achieve the same level of diagnostic efficiency and accuracy after the biopsy of one cell as compared to two-cell biopsy.
In order to answer these questions we enrolled patients with embryo biopsy for PGD or PGS in a randomized controlled trial (RCT) and assessed embryonic development, delivery rate with live birth and clinical outcome on all cycles, as well as the diagnostic efficiency after the removal of one or two blastomeres of both PCR and FISH techniques for PGD. These primary outcomes were analysed by cycle. As a secondary outcome, diagnostic accuracy, more specifically false-positive rate per embryo reanalysed, was assessed only for the PCR cycles.
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Materials and Methods |
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Patients and cycles
Between May 2001 and September 2005, 592 cycles (106 PCR and 486 FISH cycles) were included in the RCT. Inclusion criteria were that a duplex- or multiplex PCR was available for the monogenic diseases or FISH was performed for sexing, chromosomal abnormalities or aneuploidy screening. PGD cycles for which only simplex PCRs were available or in which structural chromosomal abnormalities had to be detected were excluded from the study. The person responsible for embryo biopsy assigned biopsy cycles to one-cell or two-cell biopsy in the morning of Day 3, according to a computer-generated randomization list (blocks of 200 cycles), concealed for allocation (Fig. 1). The different indications for PCR and FISH are summarized in Table I.
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Ovarian stimulation, ICSI procedure and evaluation of embryonic development
Two ovarian stimulation protocols were used in the present study. Initially, the long GnRH agonist protocol using Buserelin (Suprefact; Hoechst, Frankfurt, Germany) combined with HMGs (Menopur; Ferring Pharmaceuticals A/S) was applied (Van de Velde et al., 1998
). A combination of GnRH antagonist and recombinant gonadotropins was introduced in turn, and gradually replaced the long agonist protocol. The stimulation protocol was described by Kolibianakis et al. (2004).
ICSI was performed in all cycles to avoid unexpected fertilization failure and to prevent contamination with sperm adhering to the zona pellucida (ZP) (Van Landuyt et al., 2005; De Vos and Van Steirteghem, 2005). Ejaculated sperm was used for injection or, in cases of Congenital Bilateral Absence of the Vas Deferens, testicular sperm was obtained by fine needle aspiration or open excisional biopsy. After 16–18 h, fertilization was assessed and further development of the embryos was evaluated on a daily basis. For embryo evaluation on Day 2 and 3, the developmental stage was registered and the embryos were further classified as type A, B or C according to the percentage of fragmentation (Van Landuyt et al., 2005). On Day 4, embryos were scored as compacting (C1, cell borders are still visible), compact (C2, a dense morula is formed) or early blastocysts (Bl1–Bl2). Early compacting embryos on Day 3 were scored similarly. Day 5 blastocyst evaluation was done as described by Gardner and Schoolcraft (1999). Early (artificial) hatching of the blastocyst through the biopsy opening in the ZP was recorded as stage 7. Occassionally, collapsed blastocysts were observed (recorded as stage 8). Blastocysts 1 and 2 were considered as early blastocysts, good quality blastocysts included all other stages (3–8, at least type BA or BB) and top quality blastocysts included stages 3–8, types AA or AB). In the typing, the first letter refers to the quality of the inner cell mass and the second letter refers to the quality of the trophectoderm. Embryos of moderate quality included compacting embryos (still visible cell borders) and blastocysts with very few cells in the inner cell mass and/or a poor trophectoderm. Embryos of poor quality included embryos presenting partial compaction (not all cells participating) and/or few cells compacted and/or presence of additional fragments. Blastocysts without visible inner cell mass, fully fragmented embryos and degenerative embryos were also considered as embryos of poor quality. Arrested embryos are the ones showing no further development between Days 4 and 5.
Embryo biopsy and genetic analysis
Embryos of grade A, B or C with at least six blastomeres were biopsied in the morning of Day 3 after injection as described previously (Joris et al., 2003). Initially embryos were decompacted prior to biopsy by short incubation (5–10 min) in Ca2+- and Mg2+-free bicarbonate buffered medium (EB-10, Vitrolife, Kungsbacka, Sweden). Embryos were then returned to Ca2+- and Mg2+ containing HEPES buffered medium for biopsy. Whenever HEPES buffered Ca2+- and Mg2+-free became available (EB-10 and later G-PGD, Vitrolife), decompaction and biopsy were done at the same time (embryo exposure never exceeding 5–10 min). Laser technology was used to create an opening in the ZP (Fertilase, MTM Medical Technologies, Montreux, Switzerland or Octax Laser Shot, Octax Microscience GmbH, Germany using Octax Eye Ware software). One or two-nucleated blastomeres were then gently aspirated through the hole by means of an aspiration pipette (inner diameter 35–40 µm). All biopsy practitioners were equally proficient at performing the technique. After biopsy, the embryo was transferred to the sequential medium for blastocyst culture, leaving the blastomeres in the biopsy dish. Further procedures at the blastomere level were then performed using a stereomicroscope. For PCR, each blastomere was washed three times in (home-made) Ca2+- and Mg2+-free medium [14 mM NaCl, 0.2 mM KCl, 0.04 mM NaH2PO4.2H2O, 5.5 mM glucose, 1.2 mM NaHCO3, 0.02 mM EDTA and 0.01% (w/v) phenol red] and transferred under visual control to a 0.2 ml PCR tube containing 2.5 µl alkaline lysis buffer (50 mM dithiothreitol and 200 mM KOH or NaOH). They were kept at –80°C for at least 30 min (Sermon et al., 1998) prior to the lysis and PCR. For sex determination, detection of chromosomal abnormalities as well as aneuploidy screening, the blastomeres were spread on a slide according to the HCl/Tween20 method and FISH procedures were carried out as previously described (Staessen et al., 2003). Some PCR procedures and primers have been published before (De Vos et al., 2003; Goossens et al., 2003; De Rycke et al., 2005; Spits et al., 2005, 2006). Other primer sequences are available on request.
Diagnostic efficiency of PCR and FISH PGD cycles
For the analysis of the efficiency of the diagnosis, PCR cycles and FISH PGD cycles were considered separately. FISH PGS cycles were not considered (see Michiels et al., 2006). This primary outcome parameter has been analysed by cycle. Embryos for which the number of cells removed differed from the number that was indicated in the randomization table were excluded from the analysis. These included embryos where an extra cell was removed due to cell lysis, absence of a nucleus or presence of multinucleation as well as embryos where only one cell was taken instead of two due to poor quality of the embryo. Therefore, the diagnostic efficiency represents the proportion of embryos that could be diagnosed per cycle after biopsy of one cell in group I or two cells in group II. Embryos without diagnosis were further stratified according to the reasons underlying the failure of diagnosis.
Diagnostic accuracy of the PCR cycles (reanalysed embryos)
Affected embryos, embryos without a diagnosis or genetically abnormal embryos as well as embryos that were genetically normal for the test being performed, but not transferred or frozen, were reanalysed with written consent from all patients to assess the diagnostic accuracy, and more specifically, the false-positive rate. Each embryo, obtained on Day 5 or 6, was analysed as a single sample (after the removal of the ZP), using the same PCR protocol as during the PGD cycle. The diagnosis was considered as confirmed if the same diagnosis was obtained as during the PGD (affected/affected, genetically normal for the test being performed/genetically normal for the test being performed,...), and as not confirmed if during the PGD no diagnosis could be given and post-PGD a result was obtained (no diagnosis/genetically normal for the test being performed,...) or vice versa. The diagnosis was considered wrong if during the post-PCR a different diagnosis was given than during the PGD (genetically normal for the test being performed/affected, i.e. false negative, affected/genetically normal for the test being performed, i.e. false positive,...). It is not possible to report the standard diagnostic accuracy (sum of true negatives plus true positives over the total), given that false-negative embryos may have been transferred. Only false-positive rate (number of healthy post-PGD results per number of affected/abnormal embryos reanalysed) is reported as a secondary outcome. For false-negative rate estimation, fewer embryos were available for reanalysis (majority of them transferred or frozen).
Clinical outcome
Two increasing hCG concentrations >10 IU/l at least 10 days following embryo transfer were considered as positive. The implantation rate represents the ratio between the number of gestational sacs with a fetal heartbeat and the total number of embryos transferred. A clinical pregnancy was defined by the presence of an intrauterine gestational sac with positive fetal heart activity at ultrasound performed at least 7 weeks post-embryo transfer. Follow-up data on pregnancies and children were prospectively collected via questionnaires given to the patients at the day of transfer and sent to the patients and their physicians (gynaecologist, paediatrician) during pregnancy, at delivery and thereafter. Details of pre- and post-natal diagnosis results are included in the Supplementary data, as well as details on the children born and on congenital malformations and neonatal complications at birth. Whenever possible, children were examined at 2 months and at 2 years of age.
Statistical analysis
Considering the delivery rate with live birth as primary endpoint (expected to be around 17% based on a large data collection in literature, Harper et al., 2006), a sample size calculation for a difference of 8% resulted in 283 cycles per treatment group.
For variables of interest, comparison between group I (one cell removed) and group II (two cells removed) was done by generalized estimating equations (GEE), which allows general linear modelling for repeated measures (Liang and Zeger, 1986). Cycles from a same patient were considered to be exchangeable as well as embryos from a same cycle. A logistic model was fitted to the binary outcome variables, for continuous outcome variables normal regression was used. To evaluate the influence of the intervention on the outcome variables, the P-value associated to the likelihood ratio statistic for the intervention effect is reported. Results were considered significant if P < 0.05 (
= 5%) for the primary outcome. Tables with descriptive statistics show the least square mean and its SE computed by the GEE model for continuous outcome variables and mean percentages per cycle for binary outcome variables. These mean percentages can differ from the percentages calculated as total number of events over number of cycles.
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Results |
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Evaluation of embryonic development
According to the randomization list, the cycles were divided into two groups: in the first group (group I) only one blastomere was removed for analysis (288 cycles, 52 PCR cycles, 236 FISH cycles) and in the second group (group II) two blastomeres were biopsied and analysed (304 cycles, 54 PCR cycles, 250 FISH cycles).
All characteristics of group I and group II cycles, including the number of women participating in the study, the numbers of injected and fertilized oocytes, and numbers of embryos biopsied in the two groups are summarized in Table II. There was no statistical difference for any of these parameters.
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The developmental stage and morphological quality of the biopsied embryos in the two groups on Day 3 (day of biopsy) were compared (Table III). Both groups represented a similar embryo population for biopsy in which >60% of the embryos had at least eight blastomeres or were already compacting or compact. The majority of the embryos were of good (grade B) to excellent (grade A) quality: 79.7% (n = 1380) of the embryos had <20% fragmentation in the group I, compared with 82.2% (n = 1402) in the group II. It should be noted that fragmentation was not scored in compacting/compact embryos. Statistical analysis by GEE modelling showed that the morphological parameters (developmental stage and fragmentation in the embryos) did not differ significantly (P > 0.05) between group I and group II on Day 3.
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Blastocyst formation was evaluated in the morning of Day 5. One hundred and thirty-six embryos were not scored on Day 5 because they were reanalysed on Day 4 for logistic reasons. In group I, the overall blastocyst formation rate was 829/1608 (51.6%) of the biopsied embryos. The overall blastocyst formation rate for group II was 767/1633 (47.0%) of the biopsied embryos.
The influence of Day 3 developmental stage (as a biological parameter) and/or the influence of one- or two-cell biopsy (as the intervention in itself) on Day 5 blastocyst formation are shown in Table IV and Fig. 2. Compacting/compact embryos on Day 3 were not included in this analysis because of the inability to score their cell stage (which may be extremely variable). GEE modelling showed that the in vitro development on Day 5 was mainly influenced by the embryonic development on Day 3, i.e. that the main parameter determining the quality on Day 5, is the cell stage on Day 3: a clear statistical difference could be demonstrated (P < 0.0001). The removal of one or two cells also has a significant influence on embryo development on Day 5 (P = 0.007). The interaction between both parameters, Day 3 developmental stage and one- or two-cell biopsy, was only significant if early, good and top quality blastocysts were grouped together (P = 0.026).
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Diagnostic efficiency of PCR and FISH PGD cycles
For the 106 PCR cycles, group I consisted of 52 cycles or 330 embryos and group II consisted of 54 cycles or 329 embryos for diagnosis (Table V). The PCR cycles can be divided into two groups, the autosomal and X-linked recessive and the autosomal and X-linked dominant disorders (Table I). For the recessive disorders, three out of four embryos are genetically transferable and these embryos are homozygous or hemizygous healthy or heterozygous carrier embryos, free of the disease under consideration. For the dominant disorders only one out of two embryos is genetically transferable or homozygous healthy. In total, for group I of the PCR cycles, 35 (11.4%) of the 330 biopsied embryos were lost for transfer, because no diagnosis could be established. In group II, only 13 of 329 embryos (3.6%) remained without a diagnosis. The absolute risk reduction (ARR) was 7.7%, P = 0.006. Thus, for every 13 PCR PGD cycles there will be one fewer embryo with ‘no diagnosis’ if two cells are biopsied rather than one [95% confidence interval (CI): 8, 41]. Statistical analysis showed a significant difference in diagnostic efficiency calculated by cycle between group I, 88.6% and group II, 96.4% (P = 0.008).
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Lack of diagnosis in 35 out of 330 embryos (group I) where one cell had been removed was mainly due to non-amplification (22 embryos; 6 in which only one locus was amplified and 16 with complete amplification failure). Other reasons involved ADO (seven embryos), uninterpretable results (five embryos) and contamination (one embryo). In group II, 13 embryos remained without a diagnosis for the following reasons: no amplification of the two cells (5 embryos), ADO (4 embryos), uninterpretable results (3 embryos) and contamination (1 embryo). Overall, in group I ADO was found in 20 out of 314 blastomeres analysed. These 314 blastomeres represent 395 heterozygote loci of which 22 showed ADO (5.6%). In the group II comparable results were found: ADO was found in 38 out of 614 blastomeres, representing 46 out of 881 heterozygote loci (5.2%).
Embryos without diagnosis are lost for transfer. However, this loss should be put into perspective. Of these 48 embryos without diagnosis, 37 were evaluated on Day 5 showing 23 embryos with (moderate, poor, arrested) morphology (of little value for embryo transfer), 5 compact embryos, 8 early blastocysts and 1 good quality blastocyst. The overall transfer rate for the PCR cycles was 80.0%. This was not different for the cycles where no diagnosis was encountered in one or more embryos (81.3%) (data not shown). It should be added that in 32 embryos of the group II the diagnosis was based on a single amplified cell, but relying on at least two loci. Evaluation on Day 5 of this subgroup (30 embryos assessed) showed 17 embryos with (moderate/poor/arrested) morphology, which were excluded from embryo transfer; 3 compact embryos and 10 embryos belonging to the (top, good, early) quality group.
For the 78 FISH PGD cycles, group I contained 43 cycles or 259 embryos and group II consisted of 35 cycles or 177 embryos. For the group where only one cell was removed, only five embryos (1.8%) remained without a diagnosis. For the group where two cells were removed no diagnosis could be reached in four embryos (2.5%). Statistical analysis showed no significant difference in diagnostic efficiency calculated by cycle between group I, 98.2% and group II, 97.5% (P = 0.838).
Lack of FISH diagnosis was due to the fact that no FISH could be performed, mainly because no nucleus was found at the fixation of the biopsied cells (three out of four embryos in group II). Other reasons why no diagnosis was obtained were uninterpretable results (four embryos in group I) and the absence of fluorescent signals (one embryo in group I and one embryo in group II).
Diagnostic accuracy (false-positive rate) of PCR cycles
In a next step the accuracy of the PCR cycles, i.e. the occurrence of misdiagnoses, when analysing only one cell versus analysing two cells was examined. A successful reanalysis was obtained in 154/190, i.e. 81.1% (group I) and in 168/189, i.e. 88.9% (group II) embryos while in 36 and 21 embryos, respectively, reanalysis failed. Estimating the false-positive rate on reanalysed affected/abnormal embryos gave 0/91 (0.0%, group I) and 6/97 (6.2%, group II).
Clinical outcome
The clinical results of this prospective RCT are summarized in Table VI. Thirteen and five started cycles in group I and group II, respectively, did not result in an embryo transfer due to the fact that no genetically normal embryos were available. Insufficient embryo quality on Day 5 was more prevalent in group II as a reason for no embryo transfer than in group I. However, the overall transfer rate per started cycle with embryo biopsy was not different between the two groups: 77.1 and 75.3%, respectively (GEE modelling, P = 0.723). The mean number of embryos replaced per transfer cycle was not different (GEE modelling, P = 0.810). The percentage of positive hCG per started cycle was close to significance (P = 0.068), however, implantation rates, expressed as positive fetal heart beat at 7 weeks per embryo transferred, were similar in group I and group II: 23.5 and 17.3%, respectively (GEE modelling, P = 0.216). In group I, there were 58 deliveries with live birth and in group II there were 52. At the level of live births per started cycle, no significant difference between the two groups was observed (20.2 and 17.2% respectively, GEE modelling, P = 0.358). The ARR for deliveries with live birth was 3.05% (95% CI: –3.24, 9.34).
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In group I and II 68 and 61 children were born, respectively. Two babies were stillborn in each group: in group I, a twin died in utero at 25 weeks (the other twin was live born at 37 weeks) and another baby was stillborn with major malformations; in group II one baby was stillborn after abruption placentae and for one stillborn baby no apparent reason could be found. One severely premature twin (group I) died in the neonatal period. Twenty-five of the concepti in group I and 31 in group II were checked pre- or post-natally. No abnormalities related to the diagnosis were uncovered. One abnormal result in the two-cell group was detected after amniocentesis, while three abnormalities (one in group I, two in group II) were found at ultrasound. These resulted in three elective terminations and one elective reduction. Details on pre- and post-natal diagnosis results as well as data collected on birth weight, birth length and head circumference on congenital malformations and complications at birth have been presented as Supplementary data. Because of the small sample size in both groups, no statistical analysis was performed. However, at face value, both groups were quite similar and comparable to what has been reported previously by the ESHRE PGD Consortium (Sermon et al., 2007
). No malformation seemed to be more frequent than expected, and neonatal care was mostly related to twin pregnancies. |
Discussion |
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Our RCT data show that the delivery rate with live birth per started cycle is not significantly different after two-cell biopsy than after one-cell biopsy. There would have to be 33 cycles with PGD procedures before there would be one more delivery with live birth with one-cell than with two-cell biopsy. Taking into account, the heavy physical, emotional and financial burdens laid on the couples by all the aspects of PGD treatment, the diagnosis should be as accurate and efficient as possible in order to ensure the greatest chance for a pregnancy coupled to the lowest risk for misdiagnosis. It is generally considered that the best moment to remove one or two cells from human embryos is between the six- and ten-cell stage, because at that moment all the cells are considered as totipotent, compaction has in most embryos not yet occurred, and sufficient time is left for diagnostic procedures before transfer on Days 4 or 5. However, it is not clear whether and to what extent the random removal of one or two cells from this stage may interfere with early development and compromise the implantation potential of the embryo (Hardy et al., 1990
; Edwards and Beard, 1997; Magli et al., 2004). This is why we started an RCT observing the in vitro development (until Day 5) of the embryos biopsied on Day 3, with one or two cells removed and this in combination with the diagnostic efficiency (per cycle) and accuracy (per embryo reanalysed). Clinical outcome parameters were also included, up till delivery with live birth rate per started cycle as a primary endpoint.
We demonstrated that a comparable cohort of embryos was included in the one-cell and two-cell group, irrespective whether they belonged to the PCR or the FISH group. About two-thirds of the embryos contained at least eight cells or were compact in both groups. Most embryos were of good or excellent quality. No statistical difference between the two groups was found and thus no bias was introduced this way.
The intervention of removing one or two blastomeres on Day 3 had a significant influence on Day 5 embryo quality, especially for embryos at the 6-, 7- and 8-cell stage on Day 3. Removing one blastomere was less invasive than two-cell removal, thus resulting in more top quality, good quality and early blastocysts on Day 5. However, the Day 3 developmental stage represented a stronger predictor for further development. It is mainly the quality of the embryo on Day 3 in terms of developmental stage that determines its quality on Day 5 (Langley et al., 2001). Using good to excellent quality eight-cell embryos on Day 3 gives a higher chance of reaching top quality, good quality and early blastocysts on Day 5. On the contrary, six-cell embryos on Day 3 are mainly destined to remain moderate, poor or arrested on Day 5. In our hands, those embryos are rarely of good enough quality to be transferred on Day 5. Knowing that the pregnancy and implantation rates using top quality blastocysts, for transfer on Day 5, is substantially higher than when using less good quality blastocysts (Gardner et al., 2000), the value of six-cell embryos for genetic analysis could be questioned, especially if two cells are needed for diagnosis.
For the PCR cycles, a clear difference was demonstrated in diagnostic efficiency between one-cell (88.6%) and two-cell biopsy (96.4%). For every 13 PCR PGD cycles there will be one fewer embryo with ‘no diagnosis’ if two cells are biopsied rather than one (95% CI: 8, 41). The difference in diagnostic efficiency between groups I and II does not seem to influence the transfer rate. This is related in the first place to the fact that other embryos with a diagnosis and of good morphology are available within the cycle and secondly to the fact that the group of embryos without diagnosis reflects a group of lower quality embryos with limited contribution to a good quality embryo transfer. The main reason for the lack of diagnosis is the total amplification failure of the DNA content of the cell (46 versus 38% in the one-cell and two-cell group, respectively). It is clear that the chance of removing one cell in which amplification technically fails is higher than the chance of removing two of these cells. If two-cell biopsy is performed, in most cases the second cell will still give a result and thus a diagnosis of the embryo. Amplification also fails in blastomeres of inferior quality where the DNA content is degrading. We noticed that the majority of the embryos remaining without diagnosis and many of the embryos from the two-cell group in which the diagnosis had been based on the results of a single cell represent lower quality embryos. They are not recognized at the time of biopsy because their morphological quality on Day 3 is similar to the overall group; their inferior quality becomes only apparent by Day 5, as witnessed by failed analysis and moderate to poor or arrested embryonic development.
For the FISH PGD cycles, we could not demonstrate a difference in efficiency of the diagnosis between the group where only one blastomere was removed (98.2%) and the group where two blastomeres were removed for analysis (97.5%). It seems that for the FISH PGD cycles, there is no need to biopsy two cells, since it does not increase the chance to obtain a diagnosis. Michiels et al. (2006), revealed a significant difference in proportion of embryos without diagnosis when one or two nuclei were available for FISH analysis (4.1 versus 1.8% without diagnosis when, respectively, one and two cells were analysed). It is important to notice that the approach of both studies is very different. In the retrospective study of Michiels et al. (2006) the starting point was whether there were one or two nuclei available for analysis, not the number of cells removed from the embryo, which might be a higher number if anucleated blastomeres were encountered. The current study is a prospective study starting with one or two biopsied cells. When no nucleus is found in these cells, the embryo remains without diagnosis but will be included in the study. Since both approaches are so different it seems justified to let the results exist next to each other.
Reanalysis on Day 5 or 6 of the embryos that were not transferred or frozen was done routinely in the PCR cycles included in the study to confirm the results obtained on Day 3. There were six false positives, all in the two-cell group. This leads us to 0% in the one-cell group and 3.4% in the two-cell group of embryos in which the original diagnosis during the PGD cycle was not confirmed in a post-PGD check. This is because the diagnosis of affected is more readily assigned even if there is some doubt. False negatives were not found, although it can be argued that these embryos may have been transferred. If they implant, they may be identified during prenatal diagnosis, at birth or later. No abnormalities related to the diagnosis were found pre- or post-natally. Results for the FISH cycles concerning the error rates of a diagnosis based on one or two cells were already published (Michiels et al., 2006) and no false-negative results (unacceptable misdiagnoses) were found in either group. Regarding the false-positive results (acceptable misdiagnoses), an apparently increased rate was obtained; 26.6% if the diagnosis was based on one cell versus 13.6% when two cells were analysed, but this was not significantly different. Other authors have found error rates in the same range (Magli et al., 2001; Munné et al., 2003a).
It has been postulated, in studies performed on small numbers of embryos (Baart et al., 2004) or in retrospective studies (Van de Velde et al., 2000) where two cells were removed from the better quality or more rapidly developing embryos only, that the in vitro development of these embryos is not impaired when one or two blastomeres are removed. The present prospective study does reveal a significant influence of the intervention (one-cell or two-cell removal) on blastocyst formation rate and quality. The influence of developmental stage at the moment of biopsy, on the other hand, is an even stronger predictor of post-biopsy development. Interestingly enough, however, insufficient embryo development on Day 5 as a reason for embryo transfer cancellation was more prevalent in group II than in group I. This observation did not coincide with a significant difference in positive hCG per started cycle. Also, these significant in vitro developmental differences post-biopsy between the two groups did not result in a significant difference in implantation potential of the embryos transferred. The implantation rates obtained in this study correlate well with reports in literature, ranging from 11 to 18% (Vandervorst et al., 2000; Munné et al., 2003b; Staessen et al., 2004; Gianaroli et al., 2005; Feyereisen et al., 2007).
The present prospective study assessed the policy of one-cell or two-cell biopsy, taking into account not only the efficiency and accuracy of the diagnosis, but also the impact on embryonic development and thus clinical outcome, where delivery rate with live birth was used as endpoint. No significant difference was obtained at this level. However, critical interpretation of the data reveals an absolute difference in delivery rate with live birth of 3%. So for every 33 cycles, there will be one more delivery with live birth with one-cell than with two-cell biopsy (95% CI: –31, 11). Therefore, one-cell biopsy is recommended in these cases where other safety measures are in place to ensure an accurate diagnosis (multiplex PCR, multiple loci FISH analysis). Efficiency of PCR diagnosis is significantly lower in case of one-cell biopsy. Because non-amplification is the major cause of diagnosis failure, one-cell biopsy should carefully aim for a clear single-nucleated cell. Two-cell biopsy should be restricted for these cases where one blastomere is not compatible with an accurate diagnosis (if no multiplex PCR test is available, translocations).
To conclude, the data of this study provide evidence that for the PCR cycles the diagnostic efficiency is increased when two blastomeres are removed for analysis and that this is significantly different from the analysis of only one cell, mainly due to non-amplification of some cells. However, since the data show that in every 33 cycles, there will be one delivery less following removal of two blastomeres, we recommend removal of only one blastomere provided methods are in place for accurate diagnosis.
For the FISH PGD cycles, no increase of the efficiency can be demonstrated when removing two cells. The false-positive rate observed for the PCR cycles was not higher in case of one-cell biopsy. No false negatives were encountered, albeit on a limited number of embryos, genetically normal for the test being performed and available for reanalysis or on a limited number of concepti that were checked pre- or post-natally.
Day 5 in vitro development is clearly influenced by one-cell or two-cell embryo biopsy. However, developmental stage on Day 3 represents a stronger predictor. Despite differences for in vitro development (removal of two blastomeres significantly decreases the likelihood of blastocyst formation, compared with removal of one blastomere) between group I and group II, the implantation rate of the embryos was not significantly different. Likewise, similar delivery rates with live birth were obtained in the two groups.
Taking into account the fail safes described above, and the fact that single blastomere biopsy and analysis represents a lower work burden, we consider one-cell biopsy as a valid alternative for two-cell biopsy.
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This work was supported by grants from the Funds for Scientific Research Flanders (FWO-Vlaanderen), the University Research Council and by the Alphonse and Jean Forton Fund. V.G. is a predoctoral fellow, M.D.R. and K.S. postdoctoral fellows supported by the FWO-Vlaanderen.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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The authors wish to thank the medical, paramedical and technical staff of the Centres for Reproductive Medicine and Medical Genetics for their help and commitment. Professor John Collins is acknowledged for his statistical advise and critical review of the manuscript. The trial registration number (ISRCTN) is: ISRCTN20762192 [controlled-trials.com] .
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The first three authors have equally contributed. 
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Submitted on May 8, 2007; resubmitted on August 13, 2007; accepted on September 20, 2007.
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