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  • 1. TEMPORARY COMMITTEE ON HUMAN GENETICS AND OTHER NEW TECHNOLOGIES OF MODERN MEDECINE HEARING on 27 March 2001 Professor Joep GERAEDTS Prof. Dr. Joep Geraedts Universiteit Maastricht PO Box 1475 6201 BL Maastricht The Netherlands tel.: 00-31-43-3875840 fax: 00-31-43-3877877 e-mail: Professor Joep P.M. Geraedts (1948) studied general biology at the Catholic University of Nijmegen. In 1975 he obtained a PhD from the University of Leiden with his thesis: 'Constitutive heterochromatin as a marker for chromosomal studies in human somatic cells and spermatozoa'. From 1972-1982 he held posts as a scientist at the Institute of Human Genetics at Leiden University. In October 1982 he was appointed full professor of genetics and cell biology at the University of Maastricht. In January 1983 he became the first director of the Clinical Genetics Centre serving South-East Netherlands. He has also started the centre for preimplantation genetic diagnosis in Maastricht, which is still the only PGD-centre in the Netherlands. He has served as chairman of the Dutch Society of Human Genetics and is currently chairman of the Society of Dutch Clinical Genetics Centres. At the European level he is actively involved in the European Society of Human Reproduction and Embryology, in particular as coordinator of the Special Interest Group on Reproductive Genetics and steering member of the so-called ESHRE PGD Consortium. DV435224EN.doc EN EN
  • 2. Presentation summary PREIMPLANTATION GENETIC DIAGNOSIS IN EUROPE Preimplantation Genetic Diagnosis (PGD) is an alternative to prenatal diagnosis for the detection of monogenic and chromosomal disorders. After IVF or ICSI one or two blastomeres are biopsied from about 8-cell preimplantation embryos and tested using PCR or FISH. Unaffected embryos are selected for transfer to the uterus, preventing termination of pregnancy after prenatal diagnosis. Legislation, regulation and service of PGD varies among European countries. In 1997, the ESHRE (European Society of Human Reproduction and Embryology) PGD Consortium was formed as part of the ESHRE Special Interest Group on Reproductive Genetics, in order to undertake a long-term study of the efficacy and clinical outcome of PGD. In December 1999, the first PGD Consortium report was published discussing referrals on 323 couples, 392 PGD cycles and 82 pregnancies. In the second data collection round, contributing centres were asked to send in data from their PGD activities before this date, as well as from 1 October 1998 until 1 May 2000, in order to have as complete a review as possible of PGD practices in these centres. The cumulative data consist of 886 referrals and 1319 PGD cycles. Many couples have had affected children and have objections to (more) terminations of pregnancy. The pregnancy rate is less than 20 %. This figure is below expectancy and results from the increased maternal age and limited number of embryos available for transfer. The rate of multiple pregnancies is high. There is no indication that the children born show more abnormalities other than from multiple pregnancies. The most serious problem is misdiagnosis. Although not all of these data are encouraging, the practice of PGD is becoming more and more established and more and more different applications are emerging. Introduction Most couples having a child or a family member with a hereditary disease and having an increased risk themselves, in the recent past have had the following alternatives to decrease the risk: (1) to refrain from having children and eventually opt for adoption, (2) to accept the risk, (3) to opt for donor insemination or IVF using donor oocytes, or (4) to undergo prenatal diagnosis with or without selective abortion. Preimplantation genetic diagnosis (PGD) is an alternative option especially with regard to prenatal diagnosis. PGD takes place between fertilisation and implantation, but in most cases on the third day of early embryonic development. To obtain access to this early development stage, IVF or ICSI is necessary, although the women undergoing this treatment are normally fertile. The laboratory studies need to be conducted on minimal amount of biological material, which means biopsy of 1 or 2 cells (blastomeres) out of the 8 cells which are normally developed at day 3. With special techniques it is possible to study these cells. FISH (fluorescent in site hybridisation) is used to detect the sex and chromosomal abnormalities[1,2]. PCR is used to diagnose autosomal dominant, autosomal recessive and X-linked monogenic disorders [3]. 2/13 DV435224EN.doc EN
  • 3. Since the first report on clinically applied preimplantation genetic diagnosis (PGD) in 1990 by Handyside and colleagues in the UK [4], the number of centres involved in PGD, as well as the number of PGD treatments has increased year by year. In 1997 the ESHRE (European Society for Human Reproduction and Embryology) PGD Consortium was formed as part of the ESHRE Special Interest Group on Reproductive Genetics. The prospective and retrospective collection of data on availability, accuracy, reliability and effectiveness of PGD has been one of the major aims of the ESHRE PGD Consortium. In December 1999, the first PGD Consortium report was published discussing referrals on 323 couples, 392 PGD cycles and 82 pregnancies [5]. In the second data collection round, contributing centres were asked to send in data from their PGD activities before this date, as well as from 1 October 1998 to 1 May 2000, in order to have an as complete as possible overview of PGD practices in these centres. These data have been published recently [6]. Availability The availability of PGD in Europe has been summarised by Viville and Pergament in 1998 [7]. A more recent overview representing the situation in September 2000, is given in Table 1. From this it is clear that a division can be seen between countries with and without legislation regulating PGD. Some of the countries with legislation have a law allowing PGD (The United Kingdom, Spain) while it is clear that PGD is not allowed in others (Austria). In Germany the state of affairs is unclear since the law is interpreted in different ways. In countries without legislation PGD is sometimes allowed under the guidance of a national authority. Belgium, Greece, Italy and The Netherlands are examples of this. Referrals In total referral data have been obtained from 886 couples. The vast majority of couples have had one or more pregnancies. However, healthy children have been born in less than 25 % of them. More than a quarter of all couples have one or more affected children. Almost the same proportion of couples has suffered from spontaneous abortion or termination of pregnancy after prenatal diagnosis. This is reflected in the reasons for PGD. The most important reason is genetic risk and objection to termination of pregnancy (44 %). The group having experienced termination after prenatal diagnosis is smaller (28 %). In almost one third of the cases (29 %) the genetic indication was combined with sub- or infertility, which made IVF or ICSI necessary. If the broad indication groups are considered it is clear that chromosomal indications are becoming an increasingly important reason for referral. This is most likely a reflection of technical improvements. The referrals for monogenic disorders shows that cystic fibrosis is the most frequent reason for referral, followed by thalassemia and spinal muscular atrophy (type I) as far as the autosomal recessive disorders are concerned. The group of autosomal dominant diseases is dominated by the trinucleotide repeat disorders myotonic dystrophy (57 couples) and Huntington's disease (44 couples). For the fragile-X syndrome as well as Duchenne/Becker's muscular dystrophy 52 DV435224EN.doc 3/13 EN
  • 4. couples were referred. Referrals for several other X-linked diseases were noted. However, in most cases the numbers were small with the exception of haemophilia and Wiskott-Aldrich syndrome. In general it might be concluded that the pattern of referral indications is more or less a reflection of the genetic disorders requiring prenatal diagnosis. One of the differences that are clearly present is the number of referrals with the combination of two genetic disorders segregating at the same time. A total of 7 couples presented with this phenomenon. For the time being it will be very difficult to help these couples. In the majority of cases the patients were suitable for IVF or ICSI, and PGD was technically possible and/or ethically acceptable. For different reasons a total of 199 couples could not be accepted for PGD. Technical obstacles were the main reason for not being able to offer diagnosis. In a few cases this had to do with the fact that diagnosis on frozen embryos was requested. Some patients were referred to a centre that had the test already available. About 9 % of the patients did not fulfil the criteria for IVF or ICSI. Some patients were simply too old, others showed a high FSH. IVF was also considered risky to the mother in some cases of myotonic dystrophy and spinal muscular atrophy. One of the ethical objections was in the case of nondisclosure testing for Huntington's disease. The reasons for declining show that the largest group quits because of the burden of the procedure followed by the low success rate. Financial aspects appear to play a minor role. It is also interesting to note that in 21 cases a spontaneous pregnancy was the reason for declining. Cycles Cycle data were obtained on 1319 cycles. Screening for chromosome abnormalities was done in 465 cycles. All patients were infertile and the indications included repeated IVF failure, maternal age, and recurrent abortion. From a total of 6025 oocytes retrieved a fertilisation rate of 62 % was achieved. Seventy-nine percent of cycles resulted in an embryo transfer, although for a number of cycles no embryos were diagnosed as transferable, and embryos were still transferred. This is reflected in the data by the fact that the number of embryos transferred is more than the number of embryos transferable. The hCG levels were not recorded by one centre, but the clinical pregnancy rate was 29% per oocyte retrieval and 36% per embryo transfer procedure. All other cycles, a total of 843 cycles were started because of true PGD. About 10 %, 82 cycles, were cancelled before the oocyte retrieval due to a poor response, cysts or other reasons (9.7% cancellation rate). Seven hundred and seventy-one cycles reached the stage of oocyte retrieval. From 10267 oocytes collected, a fertilisation rate of 63 % was obtained. The number of oocytes inseminated is not an accurate figure, as some centres did not record this information. From the 6465 fertilised oocytes, 81% were suitable for biopsy, of which 96% were successfully biopsied, which is consistent with the data for last year. The majority of cases had cleavage stage biopsy (755 cycles) all of which used blastomere aspiration to remove the cell. The diagnosis was obtained in 86 % of embryos successfully biopsied, and of these, 46 % were diagnosed as suitable for transfer. From the number of oocytes collected, only 18 % were finally 4/13 DV435224EN.doc EN
  • 5. diagnosed as suitable for transfer, which confirms the need for the retrieval of a high number of oocytes for a successful PGD cycle. A total of 639 cycles reached the embryo transfer stage and 1340 embryos were transferred. In this series, 360 embryos have been cryopreserved, and some have been thawed and transferred, but there has been no publication reporting a pregnancy from frozen-thawed embryos. A positive hCG was detected in 174 cycles (23 % per oocyte retrieval) and 141 were confirmed as clinical pregnancies following an ultrasound scan (16.5 % per started cycle, 18 % per oocyte retrieval, 22 % per embryo transfer procedure). When the cycles were separated according to the diagnostic method used, the following results were obtained: PCR diagnoses were performed for a variety of autosomal recessive and dominant disorders and for sexing or specific diagnosis for X-linked diseases. For the PCR diagnosis, 385 cycles reached oocyte retrieval. It is well documented that for PCR diagnosis fertilisation should be achieved by ICSI to reduce the risk of contamination from sperm embedded in the zona pellucida, but still IVF was used in 35 cycles. A successful PCR diagnosis was obtained in 81% of embryos successfully biopsied, and 55 % were diagnosed as transferable. A pregnancy rate of 22 % per oocyte retrieval and 26 % per embryo transfer procedure was obtained. FISH was used for the diagnosis of sex for X-linked disease and patients carrying Robertsonian and reciprocal translocations. More X-linked diseases can be diagnosed by a DNA-specific test, which obviates the need for sexing by FISH. For the FISH diagnosis, 386 cycles reached the stage of oocyte retrieval. A successful diagnosis was obtained in 90 % of embryos successfully biopsied and of these only 32 % was diagnosed as suitable for transfer. This was mainly due to the high levels of abnormal embryos detected for patients carrying translocations. PGD for chromosome analysis mainly involved patients carrying Robertsonian or reciprocal translocations. From a total of 196 cycles that reached the oocyte retrieval stage, ICSI was performed in most cases, some of which were probably because of poor sperm quality due to the male carrying the translocation. One cycle had IVF and ICSI. Three cycles were cancelled after the oocyte retrieval, probably due to insufficient embryo development. Acidic Tyrode’s was used for drilling in 157 cycles. Polar body biopsy was used for three cycles and cleavage stage aspiration for 190 cycles. From the 2732 oocytes collected, 85 % were fertilised, which was higher than for other types of PGD cycles. Of these, 85 % of the embryos were considered suitable for biopsy. The embryo biopsy procedure was successful in 95 % of cases and a FISH result obtained in 90 % of embryos. Only 27 % of the embryos diagnosed were considered suitable for transfer, which was just 13 % of the oocytes collected. This reflects the high level of abnormal embryos detected in this group of patients. In 19 % of cycles there were no embryos suitable for transfer. A clinical pregnancy rate of 19 % per embryo transfer procedure and 15 % per oocyte retrieval was obtained. The low number of embryos available for transfer after PGD for chromosomal aberrations is one of the subjects of study. Pregnancies Pregnancy data were obtained on 163 pregnancies. The high rate of multiple pregnancies (31 %) was in contrast to the moderate pregnancy rate per cycle (16.5 %). Although several publications have now shown that careful selection of one or two viable embryos for transfer is effective in DV435224EN.doc 5/13 EN
  • 6. reducing multiple pregnancies, this type of selection is not easily applicable in PGD. First of all, it is still unclear to what extent biopsy of one or two cells from an embryo impairs the implantation potential. Secondly, at each PGD a cohort of embryos is diagnosed as unsuitable for transfer on genetic grounds, while this cohort could well contain the embryos with the highest implantation potential. Thirdly, PGD embryos are transferred at day three or day four, while most IVF centres now transfer embryos in regular ICSI patients at day 2 or sometimes at day 5 at the blastocyst stage, which makes comparing pregnancy rates after PGD and ICSI difficult. Clearly, the PGD Consortium data collection would be an ideal tool for investigating what selection criteria apply to embryos post biopsy. Up to May 2000, data on 163 pregnancies and 224 foetal sacs have been collected, the oldest of which date from 1993. Thirty-two of the 224 foetal sacs were lost during the first trimester, leading to 138 pregnancies, which went on to the second trimester. During the second trimester, seven pregnancies were lost, two most notably by TOP after misdiagnosis discovered at prenatal diagnosis. Five triplet and one quadruplet pregnancies were reduced to one singleton and five twin pregnancies. Hundred and thirty-one pregnancies went on normally, five of which were ongoing by May 2000, three for which no further information was available, and 123 which had delivered (85 singletons, 37 twins and 1 triplet). The reason why so few ongoing pregnancies are recorded is that centres were asked to send in pregnancy and baby data when the pregnancy was completed, either through birth, miscarriage, or other reasons. Complications of pregnancy were obtained for 95 pregnancies for which complete information was available. Complications occurred in a total of 31 pregnancies with more than one complication reported for some pregnancies. An important proportion of these complications (preterm labour, premature rupture of membranes) originated from multiple pregnancies, as is also illustrated by the rate of complications (calculated on the totality of deliveries) in singletons (22 %) as compared to twins (32 %). The incidence of pregnancy loss (subclinical pregnancies (i.e. pregnancies with a positive hCG, but no foetal heart beat), clinical abortions and extra uterine pregnancies) was 30/163 or 18.4 %, which is comparable to the 22.4 % mentioned by Wisanto et al. (1995) for ICSI with ejaculated sperm. However, caution is mandatory because the retrospective nature of the data collection may lead to underestimation of chemical pregnancies could be underreported. Reassuringly, no specific complication emerges which could be linked to PGD. Not surprisingly, a higher level of prematurity (51 % for twins vs. 9 % for singletons) and delivery by C-section (54 % for twins vs. 35 % for singletons) was seen in the twins. Babies Data reported on 162 live born children were collected. The sex ratio is heavily skewed towards female children due to the high number of girls born after sexing for X–linked diseases. The average birth weight for 145 children was 2824 g, with an average of 3206 g for 81 singletons and 2344 g for 64 twins. The average birth length was 47.5 cm (n=93) and the average head circumference was 33.2 cm (n=60). Apgar scores were good (≥ 8 at birth) in 78 out of 82 children and bad (< 8 at birth) in four children, three of which showed a good further evolution and one of which, a severely premature child, died. 6/13 DV435224EN.doc EN
  • 7. The presence or absence of malformations was available for 130 children; 121 of these showed no malformation. (Again it can be assumed that the 32 for which no information was given did not show malformations). Seven children showed non life-threatening malformations ranging from Mongolian spot to bilateral clubfoot and two children died due to severe malformations (exencephaly and chylothorax). No information on possible surgical correction is available since this type of information was not requested. The cohort of 162 children described here is very similar to a cohort of 1987 children born after 'regular' ICSI described by Bonduelle et al. [8]: 52 % and 54 % were singletons, 46 % and 41 % were twins, and 2 % and 5 % were triplets respectively. Other parameters such as birth weight were also very similar: singletons weighed 3206 g and 3220 g and twins weighed 2344 g and 2421 g respectively. Birth length and head circumference were equally similar. When we apply the definition of major malformation used in this publication (i.e. malformations that generally cause functional impairment or require surgical correction), we obtain a rate of 3/130 (bilateral clubfoot, exencephaly and chylothorax) or 2.3 %. Again, this is very close to the 2.9 % obtained by Bonduelle et al. [8]. Although the numbers are still small, an important message to emerge here is dealing with one of the first concerns of the ESHRE PGD Consortium in that PGD babies are not exposed to greater risks of neonatal problems or malformation than ICSI babies. Confirmation of diagnosis In total, 116 of the 236 foetal sacs (49 %) were examined through prenatal diagnosis. Four cases unfortunately were shown to be misdiagnosed at PGD. Two of these pregnancies (one affected with myotonic dystrophy and one with β-thallassemia) were terminated, while the two other pregnancies (one with cystic fibrosis and one male foetus after sexing for X-linked retinitis pigmentosa) went on to term. Whether this boy is affected with RP is not known. The obvious confirmation of PGD for sexing by the baby’s sex at birth is not taken into account. After PGD for sexing only one misdiagnosis (see above), which was discovered at prenatal diagnosis, occurred after preimplantation sex determination using PCR. In four early miscarriages a karyotype was obtained: two miscarriages showed an abnormal karyotype (one trisomy 16 and one mosaic trisomy 22). Although both abnormal karyotypes occurred in the FISH group, these cannot be classified as misdiagnoses since the involved chromosomes were not examined at PGD. The four misdiagnoses for monogenetic diseases shows the greater technical difficulties encountered with PCR than with FISH. One of these misdiagnoses was probably due to contamination during PCR; for the other three no explanation was given or available, although it would be interesting to know why these misdiagnoses occurred in order to prevent such events in the future, possibly through guidelines issued by the PGD Consortium. Conclusions Many couples referred for PGD have had affected children and/or have objections to termination of pregnancy after prenatal diagnosis. The parental ages of the couples are increased. The pregnancy rates are not yet in agreement with a fertile population. Most probably this results from the increased parental ages and the reduction of the number of embryos available for transfer. Misdiagnosis occurs in about 2-4% and is a serious problem. There is no evidence for DV435224EN.doc 7/13 EN
  • 8. an increase of congenital abnormalities other than from the increased rate of multiple pregnancies. Although not all of these data are encouraging, the practice of PGD is becoming more and more established and more and more different applications are emerging. 8/13 DV435224EN.doc EN
  • 9. References 1. Staessen C, Van Assche E, Joris H et al. Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis. Mol Hum Reprod 1999; 5: 382-389. 2. Scriven PN, Handyside AH and Mackie Ogilvie C. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Pren Diagn 1998; 18: 1437-1549. 3. Wells D and Sherlock JK. Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Pren Diagn 1998; 18: 1389-1401. 4. Handyside AH, Kontogianni E, Hardy K et al. Pregnancies from biopsies human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768-770. 5. Viville S and Pergament D. Results of a survey of the legal status and attitudes towards preimplantation genetic diagnosis conducted in 13 different countries. Pren Diagn 1998; 18: 1374-1380. 6. ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998. Hum Reprod 1999; 14: 3138-3148. 7. ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection II (May 2000). Hum Reprod 2000; 15: 2673-2683. 8. Bonduelle M, Camus M, De Vos A. et al. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod 1999, 14 (suppl. 1): 243-264. DV435224EN.doc 9/13 EN
  • 10. COUNTRY PGD PGD PERMITTED NUMBER OF REGULATED BY CENTRES LAW Austria + - 0 Belgium - + 1 Denmark + + 1 Finland + + 0 France + + 2 Germany + ? 0 Greece - + 1 Italy - + 2 Norway + + 0 Spain + + 2 Sweden + + 2 The Netherlands - + 1 United Kingdom + + 4 10/13 DV435224EN.doc EN
  • 11. CURRICULUM VITAE PERSONAL DETAILS Name: J.P.M. Geraedts Date of birth: 17 April 1948 Place of birth: Swalmen Address Kerkstraat 79 6267 EB Cadier en Keer Netherlands Civil status Married with 2 children EDUCATION: 1960-1965: Episcopal College Roermond Secondary school-leaving certificate 1965.1972 Catholic University of Nijmegen 04-06-1969: Degree in biology 01-02-1972: Master's in biology 05-11-1975: Doctorate, University of Leiden with a thesis on: Constitutive heterochromatin as a marker for chromosomal studies in human somatic cells and spermatozoa. 29-11-1985: Registered human geneticist (SMBWO: Institute for Medical and Scientific Research) POSTS HELD: 1972-1975: University clinic, Leiden Scientific assistant 1975-1982: University of Leiden (chief) scientific assistant 1983-present University of Maastricht Professor of genetics and cell biology DV435224EN.doc 11/13 EN
  • 12. OTHER POSTS: Stichting Klinische Genetica Zuid-Oost Nederland: Director (since 1983) Stichting Beheer Gebouw Drie-X Factoren: Vice-president (1989-1995) President (since 1995) Stichting Simonsfonds: President (1983-1989) Stichting Medisch Biologisch Wetenschappelijk Onderzoeker Member of board (1983-1989) Chairman of the Human Genetics Assessment Panel (1983-1989) Stichting Netwerk Zwakzinnigenzorg Benelux: Secretary/Treasurer (since 1991) Stichting Automatisering Klinisch Genetische Registratie: Chairman of the steering group (1989-1993) Nederlandse Anthropogenetische Vereniging: Member of board (1981-1985) President (1985-1989) VERENIGING VAN STICHTINGEN KLINISCHE GENETICA: Member of board (1993-2000) President (since 2000) Member of the negotiating committee (since 1994) Member of the health insurance advisory committee (since 1996) NWO: Member of the interdisciplinary Human Genome Analysis committee (1994-1998) Secretary, working party on chromosome and gene analysis (since 1990) Organiser of the annual retreat in Rolduc (since 1991) KNAW: Member of the Transgene Organism committee (1994-1997) Member, committee on animal experiments, transgenesis and biotechnology (since 1997) Stichting Biowetenschappen en Maatschappij: Member of board (since 1996) European Society of Human Genetics and Embryology: 12/13 DV435224EN.doc EN
  • 13. Member of Preimplantation Genetic Diagnosis consortium (since 1997) Coordinator Special Interest Group Reproductive Genetics (since 2000) Member, International Scientific Committee (since 2000) Member, International Advisory Committee (since 2000) Saudi Arabian Fertility Society: Member, International Advisory Committee (since 1998) Maastricht University Clinic: Member, IVF Committee (since 1992) RIVM: Member, 'Effects of Prevention' group of experts; 1997 health surveys University of Maastricht: Head of division, development biology, GROW research institute Chairman, research trainees committee (1992-1994) Chairman, medical faculty examination board (1994-1998) Member of examinations appeal board (since 1998) EU: Dutch member of jury, European Union Contest for Young Scientists (1995-1997) Membership of editorial boards: Medische Genetica (since 1983) Dezen en Genen (since 1989) Balkan Journal Medical Genetics (since 1997) Prenatal Diagnosis, PGD section (since 2000) Mediator (since 2000) Membership of academic bodies: Nederlandse Anthropogenetische Vereniging (since 1972) European Society of Human Genetics (since 1972) Nederlandse Genetische Vereniging (since 1979) Nederlandse Vereniging voor Oncologie (since 1981) Nederlandse Vereniging voor Celbiologie (since 1982) European Society of Human Reproduction and Embryology (since 1988) America Society of Human Genetics (since 1993) DV435224EN.doc 13/13 EN