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Abstract |
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Key words: Consortium data collection/FISH/gender screening/PCR/PDG
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Introduction |
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Materials and methods |
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Data collection
For information on the content of the different forms (referral, cycle, pregnancy, baby, biopsy protocol, FISH protocol, PCR protocol) we refer to the first PGD Consortium report (ESHRE PGD Consortium Steering Committee, 1999). Before March 2000, data were collected on hard-copy forms that were then sent to ESHRE Central Office. Since then, centres have used directly the blank Excel spreadsheets which were used by the Steering Committee members for processing of the data and which have been distributed through e-mail. All participating centres have received the complete spreadsheets after they had been corrected by the Steering Committee.
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Results |
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With respect to the reproductive histories for a total of 1561 cycles, it is clear that the majority of patients has had one or more pregnancies already (Table I), although the vast majority has no healthy children. About one-quarter of the couples has one or more affected children, while an even larger percentage of the couples have experienced spontaneous abortions or terminations of pregnancy after prenatal diagnosis.
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The most important reasons for declining remained unchanged (Table VII): inconvenience or burden of IVF or ICSI, spontaneous pregnancy and low success rate. The costs of the procedure was the reason for declining in only a few cases.
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PGD for chromosomal abnormalities
Table IXA shows the data for this year and previous years, and the cumulative data for all three years. For the first time it has been possible to break down the data for 2001 into the type of chromosome abnormality, i.e. Robertsonian or reciprocal translocation or other chromosome abnormality. This data could be further analysed, e.g. sex of the carrier, etc., and it is hoped that this will be completed in the next PGD Consortium report.
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For the reciprocal translocations, a total of 96 cycles reached the OR stage, and the average female age was 34 years. In this case, only 25 patients were infertile, which was much lower than in the case of Robertsonian translocations. ICSI was undertaken in most cases. All embryo biopsies were performed at the cleavage stage, using mostly blastomere aspiration. The majority of clinics used acid Tyrode's for zona drilling, which is also different to the cases of Robertsonian translocations. From 1570 oocytes, 1016 fertilized (65%), 866 were suitable for biopsy and 856 of these were successfully biopsied (99%). The diagnosis was successful in 789 cases (92% of embryos successfully biopsied), and 195 embryos were diagnosed as transferable (13% of oocytes collected). Seventy-three embryo transfer procedures were conducted (76% of OR), and 17 clinical pregnancies resulted (18% of OR and 23% of embryo transfer). The karyotypes of the chromosomal rearrangements for which PGD has been performed are listed in Table IXB.
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The cumulative data for all three years show that 368 cycles have now been performed, with 689 embryos being suitable for transfer (13% of the oocytes collected), 290 embryo transfer procedures and 62 clinical pregnancies (17% per OR and 21% per embryo transfer).
Sexing by FISH for X-linked disease
Almost all clinics performing sexing for X-linked disease use FISH. Table X shows the FISH results of sexing for X-linked disease and other non-specific X-linked conditions, e.g. autism. For sexing for X-linked disease in 2001, a total of 64 cycles reached the OR stage. The patients had an average age of 35 years, and only 17 cycles were for infertile patients. ICSI was used in most of the cases (n = 42). For all cycles, cleavage-stage biopsy was performed using aspiration to remove the blastomere, and equal numbers used acid Tyrode's or a laser for zona drilling. A total of 852 oocytes was collected, 535 fertilized (63%), 413 were successfully biopsied (98%) and 356 of these were successfully diagnosed and 153 considered transferable (18% of oocytes retrieved). Embryos were transferred in 57 cases, with a clinical pregnancy rate of 20% per OR and 23% per embryo transfer.
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PCR diagnosis for single gene disorders
Last year it was disappointing that the exact diseases for which some centres had performed PGD using PCR could not be evaluated; hence, only one overall table of PCR PGD was reported, without a list of diseases that had been diagnosed. Due to alterations in the data collection to ensure that centres made it clear which disease they were diagnosing, this year it was possible to present a breakdown of the data (Table XIA) and a list of diseases diagnosed (Table XIB
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For the autosomal dominant diseases, 72 cycles were started, and 69 reached the stage of OR. The average maternal age was 31 years, and only 8% were infertile. Most cycles used the laser for drilling, 64 cycles used cleavage-stage aspiration and three used cleavage-stage extrusion. From 929 oocytes collected, 604 fertilized (65%), embryo biopsy was performed on 381 embryos and was successful in all cases. In total, 324 embryos were successfully diagnosed (85%) and 154 were diagnosed as transferable (normal only)(48%). As expected, this is lower than for autosomal recessive diseases, as for dominant diseases embryos are either normal or affected. From 72 cycles started, only 55 had embryo transfer procedures (76%) which reflects the high number of cycles with only affected embryos, while 11 resulted in a clinical pregnancy (16% pregnancy rate per OR and 20% per embryo transfer procedure).
Nine cycles were undertaken for a specific diagnosis of an X-linked recessive disease, and 15 for X-linked dominant. Three pregnancies were obtained for the X-linked recessive disease and only one for the X-linked dominant. Four cycles were cancelled in the X-linked dominant group and these were all for Fragile X patients. Three cycles were performed where the patients carried two diseases; in all cases this involved an autosomal recessive and an X-linked disease. Two cycles were performed for mitochondrial diseases; in these cases polar body was combined with cleavage-stage biopsy, but no embryos were diagnosed as transferable.
The cumulative data for all three years show that there have been 575 cycles to OR, with 36 cycles having IVF (all should have had ICSI). The majority of cases used cleavage-stage biopsy with aspiration, and 479 cycles resulted in embryo transfers (83%) and 119 clinical pregnancies (21% per OR and 25% per embryo transfer procedure).
PGD-AS
The Consortium discussed which indications were now being performed for aneuploidy screening, and the following groups were identified: (i) age >35 years; (ii) recurrent IVF failure, defined as at least three failed IVF attempts (in Australia, the government pays for unlimited IVF cycles and their definition would be more than 10 embryos transferred); (iii) more than two miscarriages with the parents having a normal karyotype; and (iv) other, which would include patients with two indications, and other reasons for performing PGD-AS that do not fit into the above categories.
This year, a total of 334 PGD-AS new cycles were included in these data, from 11 centres (Table XII). For 2001, 101 cycles were performed for age alone, 45 for recurrent miscarriage with the parents having normal karyotypes, 117 cycles for recurrent IVF failure, and 71 cycles for other indications. For all indications the majority of patients were infertile. Cleavage-stage aspiration was used in all cases for 2001. Overall, only 40% of embryos were suitable for transfer, and only 75% of cycles had an embryo transfer procedure. Sixty-six cycles resulted in a clinical pregnancy (20% per OR and 26% per embryo transfer procedure). The only striking result was the low pregnancy rate for patients with recurrent IVF failure (7% per OR and 11% per embryo transfer procedure). Analysis of the spare embryos which were not transferred showed that in 8% the original result could not be confirmed. This may have been due to chromosomal mosaicism, as the majority of clinics performing PGD-AS take only one cell, but FISH failures cannot be ruled out.
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PGD for social sexing
For social sexing, the majority of patients were fertile (Table XIII). The average age was 36 years. All cycles except one used FISH for the PGD. One cycle used PCR, and from five oocytes collected, three were fertilized but no embryos were biopsied. A total of 78 cycles reached the OR stage, with the majority having IVF, laser drilling and cleavage-stage aspiration. From 1003 oocytes, 735 were fertilized, 623 embryos were biopsied, and 579 were successfully biopsied, of which 241 (41%) were diagnosed as transferable. A clinical pregnancy rate of 35% per OR was achieved, which is higher than any other PGD pregnancy rate. No information was given regarding the sex selected for.
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In 157 of the 215 pregnancies which went to delivery, data on possible complications during pregnancy were completed (Table XV). In the other cases the field `complications in pregnancy' was left blank. Presumably, there were no complications in these latter pregnancies, but for scientific correctness these blank fields were not included. Complications were present in 52/157 (33%) of the pregnancies. As expected, the number of complications was higher in the multiple pregnancies compared with singletons. It is obvious that premature contractions and premature rupture of membranes were the commonest complications and were closely related to multiplicity. Other complications observed were bleeding, hypertension and (pre)-eclampsia. These data are comparable with those obtained in the previous data collection.
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Discussion |
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Cycles
Currently, a growing number of PGD cases are being performed. This year, it was useful to have details of the patient's infertility and the female's age. However, the Consortium need to clarify what is meant by infertility, as some groups feel that patients carrying chromosome abnormalities who are experiencing repeated miscarriages are infertile. There is still one case where IVF was used in a PCR cycle instead of ICSI, but this is an improvement on last year's data. The pregnancy rates are slowly improving. The use of the laser is increasing, there are few polar body biopsies (both backed-up by cleavage-stage biopsy for mitochondrial diseases), and no blastocyst biopsies. As discussed earlier, blastocyst biopsy may not be a viable option for PGD as a large number of embryos are required. Therefore, the majority of cases used cleavage-stage biopsy, with aspiration to remove the blastomere. The debate of whether to take one or two cells for the diagnosis is still under discussion. A growing number of embryos are being frozen in PGD cycles, but no clinical pregnancies have been reported from frozenthawed embryos except one from comparative genomic hybridization (CGH) (Wilton et al., 2001). Another Australian group has reported good results for cryopreservation after embryo biopsy (Lalic et al., 2001
), while other authors have found it to be detrimental (Magli et al., 1999
).
It has been useful in this report to have a breakdown of the PCR and translocation data, and it is hoped to continue this in the future and apply this to all the collected data.
A very high number of PGD cycles was for patients carrying chromosomal abnormalities, as these patients often experience repeated miscarriages and many feel that PGD is their only hope of having a normal child (Munné et al., 2000). Current protocols using either a combination of telomeric and centrometric probes (Scriven et al., 1998
; Munné et al., 2000
) or cell conversion (Verlinsky and Evsikov, 1999
), makes this test much more simple than previous protocols. Another problem is that a high number of abnormal embryos are often found in these cases, reflected by the low number of transferable embryos and reduced number of cycles with an embryo transfer procedure. A separate publication on this matter is on the agenda of the Steering Committee.
The number of PGD cycles for sexing for X-linked disease has decreased as compared with previous years. This might be explained by the fact that, as more X-linked diseases are characterized at the molecular level, patients would opt for specific DNA diagnosis rather than simple sexing. However, the drop in sexing with FISH is not reflected in a rise of specific PCR-PGD diagnoses. It was reassuring to see that the low pregnancy rate achieved in the first report (only 7%) has now risen to 20% per OR, which is comparable with the pregnancy rate for social sexing.
As for the other FISH and PCR cycles, the centres were requested to specify the indications for PGD-AS more clearly. As this was done at a later stage in the data collection, it was still not possible to break down the data this year. In the next data report it is hoped that it might be possible to separate out those patients who have had a previous abnormal pregnancy, but this could not be done in the present report as some centres had not made this clear. The strikingly low pregnancy rate in the group of recurrent IVF failure (7%) raises certain comments. First, the Consortium here fulfils one of its roles, namely to identify problems which may not be apparent when smaller groups are considered. Second, as was emphasized by one of the members, these patients had 0% success before treatment, and even though 7% is still low when compared with other types of PGD-AS, it is still an improvement over these patients' original prognoses. In addition, many centres use this procedure to give some closure to these patients, by showing them that the reason for IVF failure is, in many cases, a lack of chromosomally normal embryos.
What can be remembered from the analysis of the PCR cycles, is that autosomal recessive diseases have a better outcome than autosomal dominant diseases. This is of course very simply explained by the fact that in the first situation 75% of the embryos are expected to be without the disease in question, while in the latter there are only 50%. This basic fact of Mendelian inheritance also explains why patients carrying an autosomal dominant disease will more rapidly turn to PGD, since their risk for an affected fetus at prenatal diagnosis is higher. This fact is even more to the point for patients who are at a combined risk for an autosomal recessive disease and an X-linked disease (in this case Fragile X). It can be calculated that these patients have one chance in four for a pregnancy affected with the autosomal recessive disease, and one chance in two for a pregnancy affected with Fragile X, leading to the chance of five out of eight that a pregnancy would be affected with either or both of the two genetic diseases. The high number of cycles which had to be cancelled for Fragile X, is altogether not surprising. It is known that carriers of premutations suffer from premature ovarian failure, and the fact that these patients are difficult to stimulate may be a reflection of this (Sherman, 2000). A new category of disease for which PGD has been performed are the mitochondrial diseases, which are especially challenging because of their complex inheritance patterns which are difficult to predict. Further results and developments for mitochondrial diseases are awaited with much interest.
For the first time, three centres submitted data after having performed PGD for social sexing. A survey among the Consortium members showed that from the 21 centres that replied, 15 were against social sexing, while four were in favour (only one of the three centres performing sexing replied). Two centres did not clearly mention their opinion. Among the arguments for sex selection, the right to self-regulation of countries was mentioned. One of the respondents, who answered in a personal capacity and not for his/her centre and who was in favour of sexing, attached a few conditions in that: (i) sexing should be used for family balancing; (ii) there should be a balance in the sex ratio within one centre and within one year (as many cases for boys as for girls); (iii) healthy embryos (i.e. embryos screened for aneuploidy as well as gender) of the unwanted sex should be donated to other couples; and (iv) patients should themselves pay for the treatment. The main argument put forward by the centre which is performing social sex selection and which responded, was that elimination of embryos of the unwanted sex was better than to perform abortion. As an argument against sexing, it was mentioned that PGD and prenatal diagnosis (PND) should only be used for serious genetic diseases and not for eugenics. One respondent also called social sexing child rights abuse, another argument was the cost to society of social sexing, and finally the influence of social sexing on the sex ratio balance was mentioned.
The Consortium members were also asked if they felt that the data from the three centres should be published in this report. Seventeen of the responding centres were in favour of publishing the data on the cycles for social sexing, and four were against. The following arguments in favour of publishing were given: (i) data on efficiency (of PGD in general) are much needed; (ii) not publishing these results would amount to censorship; (iii) PGD for social sexing should be put into context in relation to the other forms of PGD; (iv) the suppression of data would lead to much criticism if it was found out; and (v) reporting of the data should allow open debate. Some centres requested conditions to the publishing of the data. The first request was to include sex ratio at birth; this was not be possible for the current data collection, but could be added in a later data collection round. The second request (made by two centres) was to add the names of the centres performing social sexing. As the PGD Consortium collects data on an entirely voluntary and anonymous basis, this is not possible. However, as it seems that the main fear of the centres with this second request is to be associated with the centres performing social sexing, a clear statement of the general opinion within the PGD Consortium should alleviate this fear. Four centres were against publication because: (i) the publication could have a negative effect on the decision-making in their home country (or other countries where PGD is still being discussed); (ii) the PGD Consortium should not support, approve or include these centres, and hence their data should not be published; (iii) publishing these data would attract unnecessary controversy; (iv) publishing data on social sexing was akin to using data from nazi experiments; and (v) some centres do not want to be associated with social sexing. Two French centres have taken a very radical view against social sexing, and even against the publication of the results. Their statement can be found in a Letter to the Editor in this issue (Ray et al., 2001).
In conclusion, although most PGD Consortium members are against PGD for social sexing, most are in favour of publishing the data collected through the centres that are performing social sexing. As the fear was raised that social sex selection in embryos may be associated with ESHRE, the chairman of ESHRE as well as representatives from the Special Interest Group on Law and Ethics were consulted on the issue. Both agreed to the publication of results because selective withholding of data would be unethical. ESHRE has not yet issued a statement or guidelines concerning social sex selection, in contrast to the ASRM (Ethics Committee of the American Society of Reproductive Medicine, 1999), but will hopefully do so in the near future.
Pregnancies and babies
This year, there has been a shift in the indications towards more pregnancies after PGD using FISH, and this is due to the larger number of babies born after aneuploidy screening. This was to be expected as the same shift is present in the cycle data. With regard to the complications during pregnancy and at birth and the data of the children at birth, no changes can be noted compared with former data collections and comparable groups of ICSI children (Bonduelle et al., 1999; ESHRE PGD Consortium Steering Committee, 2000
). Again, the high proportion of multiplets, leading to an important number of the pregnancy complications and premature babies is something which much be warned against. This year again, a number of misdiagnoses, both prenatally and post-natally, are to be regretted. The Steering Committee feels that the time is now ripe to draw conclusions from the data gathered over 3 years, and that a start should be made with the formulation of guidelines specific for the practice of PGD.
Future prospects
This year there has been much discussion within the ESHRE PGD Consortium about two aspects of the data: social sexing (as discussed above); and the follow-up of the cycles resulting in pregnancy. It has been noted that from the first two Consortium reports, the clinical pregnancies reported in the cycle data could not be followed through in the pregnancy and baby data. This has mainly been due to centres not completing pregnancy and baby sheets for all their pregnancies reported in the cycle data. Two novelties will be introduced to eliminate this problem. First, Moutou and Viville (Strasbourg, France) have developed a database based on FileMaker Pro 5, which will allow the Consortium to collect the same data as collected hitherto, as well as an automatic calculation of results and linking of the referral sheet, cycle sheet and pregnancy sheet of one and the same patient. The second innovation is the timing at which the data will be collected. Until now, cycles for which the outcome was known as well as pregnancies and babies were collected from May to May of the next year. From now on, cycles collected during a given calendar year will only be collected in October of the next year, so that any ensuing pregnancies should be completed by the time of data collection and could also be reported at the same time as the relevant cycle. The first method allowed for a quick collection of data, which was the first aim of the PGD Consortium, while the second method will hopefully yield more complete results, albeit at a later time. Therefore, this will be the final year where the data are reported in this manner, and from next year onwards all cycles resulting in pregnancies will be completed with the data on the children born. The Steering Committee intends to spend the coming year cleaning up the existing data and filling any gaps left so that, for the next report, a more complete and accurate overview will be given of PGD and its outcome.
As the use of a more efficient database will give the Steering Committee more time, the conduction of retrospective studies will be taken up again. To this end, all members have received a list of possible studies to which they were invited to participate. We hope to be able to present the first results of these studies in the next report.
Finally, a `PGD-mail' was set up, which is similar to the broadly known `embryo mail', and which would serve the same aims, i.e. to be a forum for exchange of information and for discussion on PGD. J. Harper is currently coordinating PGD mail, and so registration to PGD mail should be addressed to her (joyce.harper{at}ucl.ac.uk).
In conclusion, the data collected this year confirmed the trends that were already apparent in the two former reports. The appearance of sex selection for social reasons has generated quite a debate within the Consortium, and we expect this to continue with the publication of this report. The Steering Committee hopes to be able to implement a series of improvements to the data already gathered and the data to be collected in the future, and ultimately to offer complete and reliable data to PGD practitioners and their patients alike.
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Acknowledgements |
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Notes |
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1 To whom correspondence should be addressed at: Centre for Medical Genetics, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail lgensnk{at}az.vub.ac.be.
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References |
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Submitted on August 2, 2001; accepted on October 4, 2001.