11th Annual Meeting on Preimplantation Genetic Diagnosis
PREIMPLANTATION GENETIC DIAGNOSIS:
EXPERIENCE OF THREE THOUSAND CLINICAL CYCLES
Report of the 11thAnnual Meeting of International Working Group on Preimplantation Genetics, in Association with 10th International Congress of Human Genetics, Vienna, May 15, 2001
Following its introduction eleven years ago, preimplantation genetic diagnosis (PGD) has become an important complement to preventive measures for genetic disorders and an option for avoiding traditional prenatal diagnosis potentially leading to abortion (International Working Group on Preimplantation Genetics 1998, 1999, 2000). The advent of PGD has also presented the opportunity for pre-selection of aneuploidy free embryos in assisted reproduction practices, improving the effectiveness of in vitro fertilization (IVF) in low prognosis patients, such as those with advanced maternal age. As reported by the previous 10th Annual Meeting of International Working Group on Preimplantation Genetics (2000), PGD has already become a generally acceptable alternative to conventional prenatal diagnosis, and applied for some conditions never previously considered as indication for prenatal diagnosis. Among other important developments noted was the introduction of the techniques for the possible full karyotyping of single blastomeres and polar bodies, which could potentially allow testing for all chromosomes to improve the pre-selection of viable embryos in assisted reproduction.
Taking into consideration the emerging importance of PGD in the current genetics and assisted reproduction practices, the present 11th Annual Meeting of International Working Group on Preimplantation Genetics was organized in conjunction with the 10th International Congress of Human Genetics held this year in Vienna. Among the major topics discussed were: (i) recent developments in full karyotyping and their current application to PGD for translocations and aneuploidy; (ii) PGD for genetic disorders combined with non-disease testing and fingerprinting; (iii) progress in the practical application of PGD to genetics and assisted reproduction practices. Resumees of these discussions are presented below.
1. Developments in full karyotyping of individual blastomeres and their application to PGD for translocations and aneuploidy
It has previously been reported that metaphase chromosomes from polar bodies and single human blastomeres could be obtained following their fusion with mouse or cow zygotes (International Working Group on Preimplantation Genetics 2000; Verlinsky and Evsikov 1999; Evsikov and Verlinsky 1999; Willadsen et al. 1999). These approaches have currently been applied in Chicago center for 38 of a total of 75 PGD cycles for translocation testing, demonstrating their high efficiency for karyotyping (Verlinsky et al., unpublished data). Of a total of 311 blastomeres tested, the technique was successful in as many as 278 (89.4%), making possible preselection of normal embryos or those with balanced chromosomal complement for transfer in 26 cycles. In addition to avoiding the use of the expensive and time-consuming customized breakpoint-spanning probes, the method was highly accurate based on the follow up testing of embryos predicted to be abnormal, and confirmatory prenatal diagnosis of the ongoing pregnancies or testing of the newborn babies. The method also allows balanced and normal embryos to be distinguished, which cannot be achieved by currently available interphase FISH analysis. Of the overall 75 PGD cycles testing for translocations, balanced or normal embryos were pre-selected for transfer in 51, resulting in 17 (30.8%) clinical pregnancies. Four of these pregnancies aborted spontaneously (23.5%), while the others resulted in healthy deliveries or are still ongoing.
Current IVF practices are gradually shifting from day 3 to day 5 embryo transfer, which allows the use of blastocyst biopsy. Karyotyping blastocysts may also have potential in the future application of PGD both for aneuploidies and translocations (Clouston and Wolstenholm, unpublished data). A series of 305 spare 5-8 day old human blastocysts has been tested for cytogenetic abnormalities, following thymidine synchronisation of cell division and cell disaggregation by 70% acetic acid. A total of 252 of these blastocysts produced metaphases, making G-banding analysis possible in 143 of them. The observed chromosomal abnormalities were in agreement with the expected types and rates, demonstrating a potential usefulness of the method for testing aneuploidies and translocations following blastocyst biopsy.
Because PGD is practically the only hope for couples with translocations to have an unaffected child without fear of repeated spontaneous abortions, increasing numbers of PGD cycles for this indication have been performed. The largest series of 134 cycles has been reported from the St. Barnabas center, resulting in 105 transfers and 42 clinical pregnancies. Overall, 207 embryos were transferred resulting in 48 fetal heartbeats (23% implantation rate). This highly positive pregnancy outcome in the series, combined with a 9-fold reduction of spontaneous abortions (9%), has to be compared to pregnancy losses (82%) in the same couples prior to PGD (Munné et al. 1998; Munné & Escudero, unpublished data).
A series of 15 PGD cycles for translocations was performed at the Guy’s & St. Thomas’ center, London, resulting in clinical pregnancies in six patients. The follow up confirmation study of the affected embryos in this series provided the material for segregation mode analysis, which demonstrated an alternate segregation in 66% of embryos resulting from female reciprocal translocation carriers, compared with 77% for Robertsonian translocations. Only 16% of embryos in the reciprocal translocation cycles were consistent with adjacent-1 segregation, the mode yielding the most common unbalanced products seen at prenatal diagnosis (Ogilvie and collaborators, unpublished data).
Of a total of 224 PGD cycles performed for translocations in the above three centers, 171 resulted in embryo transfer and 65 (38%) in clinical pregnancies, from which only one fifth has presently been done by the karyotyping method mentioned. The further follow up of the abnormal embryos, as well as outcomes of the clinical pregnancies, will be of importance to evaluate the accuracy of diagnosis. For example, in the follow up studies of the abnormal embryos cultured to blastocyst stage, mosaicism for the chromosomes involved in translocations was observed, indicating a possibility for misdiagnosis in PGD for translocations, performed by blastomere biopsy (Verlinsky and collaborators, unpublished data). This study also demonstrated that embryos with unbalanced chromosome complements have apparently the same chance of reaching blastocyst as normal embryos, explaining a high rate of spontaneous abortions in these patients (Evsikov et al. 2000).
Considerable progress is reported in the development of single cell comparative genome hybridization (CGH) technique, to enable a PCR-based full karyotyping. The method has been applied to single blastomeres, detecting a wide variety of chromosomal abnormalities, including aneuploidy and mosaicism (Wells and Delhanty 2000; Voullaire et al. 2000). In addition to detecting some unexpected class of chromosome abnormalities, the most important limitation of the present CGH protocol is still the 3-day duration of the procedure, which is incompatible with the current laboratory framework for PGD. Among approaches proposed to overcome this problem, the following are currently being explored: i) acceleration of the CGH procedure; ii) performing CGH on polar bodies; iii) embryo freezing after biopsy, with thawing postponed until after diagnosis has been completed. The latter approach has recently been applied clinically for PGD couples with recurrent implantation failures. It enabled the detection and their exclusion from transfer of as many as two thirds of the embryos tested, half of which could have been misdiagnosed as normal by the commercially available five- color probe (Williamson and collaborators, unpublished data). Progress has been also achieved in reducing a single cell CGH time to a 24-hour hybridisation, or performing CGH in polar bodies, which leaves enough time to complete CGH and transfer embryo within the implantation window (Wells, unpublished data).
2. PGD for genetic disorders combined with non-disease testing and fingerprinting
Close to one thousand PGD cycles have been performed for single gene disorders and dynamic mutations, resulting in at least 200 clinical pregnancies and 130 children born by the present time. More than half of these cases, as before, were done by the Brussels and Chicago centers, the former performing the majority of dynamic mutations using blastomere biopsy (Sermon and collaborators, 12th Fetal Cell Workshop, Prague, May 12-13, 2001, unpublished data), and the latter concentrating on single gene disorders by the polar body analysis (Rechitsky and collaborators, unpublished data).
The presently available PGD protocols provide an option not only for avoiding an affected pregnancy, but also for performing non-disease testing, such as pre-selection of the HLA compatible donors for affected siblings. Although this is still controversial in many circles, the practical application of such approach was realized in the Chicago center for HLA testing for leukemia, Fanconi anemia (IVS 4+4 A-T mutation) and thalassemia (619bp deletion). These involved an initial haplotype analysis for family members (including father, mother and affected child). This enables different polymorphic STR alleles corresponding to specific markers in HLA genes to be identified, and also tests for linked STRs scattered through HLA genes to increase accuracy of analysis and detect potential crossovers between HLA-A, HLA-B, HLA-C, HLA-E and HLA-DQB genes. Single biopsied blastomeres were tested by multiplex nested PCR analysis to analyze simultaneously for mutations, linked markers, and HLA genes. It is of interest that unaffected embryos also representing an HLA match to a sibling were detected and transferred back to patients in each of the 4 cycles performed for Fanconi anemia and 4 cycles for thalassemia mutations (1.6 embryos per cycle on the average). Sufficient numbers of embryos for transfer were also detected in both clinical cycles for leukemia, with the overall of 20 HLA matched embryos available in ten cycles, demonstrating the feasibility of preimplantation HLA matching for clinical practice. Following the first birth of an unaffected child in case of PGD for Fanconi anemia combined with HLA matching, cord blood stem cells have been collected at birth and transplanted to the affected sibling, resulting in a successful hematopoietic reconstitution (Verlinsky et al. 2001). Despite the acceptability of this new option from the medical viewpoint and that of patient autonomy, it also raised important ethical issues, which need a further consideration (Edwards, 2000; Damewood, 2001).
A combined testing for different conditions has been also performed for a couple at risk for transmitting two diseases (cystic fibrosis (CF) and fragile X syndrome). Due to the small number of available embryos for testing, only few of them free of both mutations and suitable for transfer were available, yielding no clinical pregnancies in this case (Wells and collaborators, unpublished data).
A multiplex fluorescent PCR using polymorphic microsatellite markers was introduced in the Monash center, Melbourne, for the detection of numerical changes in chromosomes using their allelic fingerprints. A tri-allelic pattern for one or more chromosome 21 markers was demonstrated to be diagnostic for trisomy 21. This allowed the development of different DNA fingerprinting systems for a wide-range application of PGD for aneuploidies and translocations, combined with mutation detection. Following a validation of this system on single trisomic buccal cells and blasmomeres from the trisomy 21 embryos detected by FISH, the method has been applied for a combined CF delta F508 and chromosome 21 fingerprinting in two PGD couples at risk for producing child with CF and Down’s syndrome. Also two new fingerprinting systems for Robertsonian translocations 13q14q and 14q21q were developed and applied in a PGD case, making possible to distinguish embryos with balanced and unbalanced chromosome constitutions (Cram and collaborators, unpublished data).
Development and improvement of multiplex PCR protocols and DNA fingerprinting will contribute considerably to the accuracy and reliability of PGD for single gene disorders and dynamic mutations. The extensive experience presently available makes it clear that misdiagnoses cannot be avoided without simultaneous amplification of the causative mutation and linked polymorphism(s). This allows the detection of allele drop out and of preferential amplification, representing common phenomena in single cell PCR analysis. For example, 116 embryos were predicted to contain the abnormal gene in a series of 55 PGD cycles performed for beta-globin gene mutations. Results showed the correct prediction of embryo genotype in 114 (98.2%), suggesting a high accuracy of PGD using multiplex nested PCR analysis (Kuliev and collaborators, unpublished data). Linked polymorphisms are particularly important for the diagnosis of dominant disorders, the accuracy of which can be severely undermined by allele dropout. To avoid misdiagnosis some centers are forced to remove two cells for testing from the day three embryos, transferring embryos on the day four, in order to maximize diagnostic accuracy (Wells and collaborators, unpublished data). Alternatively, the accuracy of PGD can be improved up to 100% by simultaneous testing for the causative mutation together with at least three linked markers (Rechitsky et al. 2000).
3. Progress in the practical application of PGD to genetics and assisted reproduction practices
Embryo or polar body biopsy has presently been applied in more than 3000 clinical cycles, resulting in an approximately 24% pregnancy rate. This is comparable to that achieved in assisted reproduction practices which do not involve biopsy of gametes and embryos. Close to 700 children have already been born following these pregnancies, with many of them still ongoing. A follow up study presently available in approximately half of these children shows 4.9% abnormalities, which is comparable to the general population prevalence, further supporting the PGD safety.
As previously reported, the procedure is also accurate, which is further confirmed by the follow up study of 435 affected embryos that were not transferred. The data indicated a 97% accuracy rate, despite a high occurrence of allele drop out in single cell PCR (Rechitsky and collaborators, unpublished data). Although no misdiagnosis was reported in PGD for translocations, embryos were identified with unbalanced translocations for other chromosomes not involved in the translocations tested, probably due to the phenomenon of inter-chromosomal effect in meiosis (Verlinsky et al. unpublished data).
In this largest PGD series from the Chicago center, a total of 1405 clinical cycles were performed, which is much larger than the series currently collected by ESHRE PGD Consortium (ESHRE Preimplantation Genetic Diagnosis Consortium, 2000). This large series allows the evaluation of clinical efficiency, safety and accuracy of the PGD practical application in a single center. The majority of these cycles (1,117) were performed for aneuploidies, the rest being applied for Mendelian diseases and translocations (see above). A total of 281 clinical pregnancies were established (23% pregnancy rate per transfer), presently resulting in the birth of 214 children, with many pregnancies still ongoing. Analysis of obstetric outcome showed a 38.8% Cesarean section (SC) rate in the term singleton pregnancies, which, however, is not different from the CS background rate in IVF patients. Although age-related aneuploidy was the major indication, the structure of indications has significantly changed in the last two years, due to an increasing number of PGD cases for Mendelian diseases and translocations. Among the indications for genetic disorders were also those that have never been observed in prenatal diagnosis, such as HLA typing (see above) and PGD for the late onset disorders, including a cancer predisposition due to p53 tumor suppressor gene mutations and Alzheimer disease (Verlinsky et al. 2001), although the further application of PGD for these considerations in still controversial. As expected, an increasing number of PGD patients presented for the second time in order to try for another unaffected child. As in previous work, the application of PGD was of special clinical significance for IVF patients of advanced maternal age, resulting in doubling of implantation and pregnancy rate in women of 39 and older (Verlinsky and collaborators, unpublished data). However, this is not a randomized controlled study, which will be needed in the future to confirm the exact clinical impact of PGD for such patients.
The other large PGD series were performed in the St. Barnabas (Livingston), SISMER (Bologna) and Brussels centers. The initial two concentrated as before on chromosomal, and the latter on genetic disorders. In addition to the above mentioned PGD series for translocations performed at the St. Barnabas center, 533 PGD cycles for aneuploidy testing were reported, to result in 191 pregnancies and 113 babies born, with 60 pregnancies still ongoing (Munne & Escudero, unpublished data). The outcome of 112 PGD cycles from this series was compared to the control cycles matched by number and age, demonstrating a marked beneficial effect of PGD for the patients of 37 years and older. The data was based on the replacement of a total of 259 embryos (with the average of 2.3 embryos per transfer) in the PGD group compared to 400 embryos (average 3.6) in the control, demonstrating an improved implantation rate following PGD (23.9% compared to 18.5% in control). The study also further confirmed previous evidence for a reduction in spontaneous abortion rates after PGD (Munne et al., 1999).
A comparable impact of aneuploidy testing was also reported from other large series, e.g. involving 457 PGD cycles performed in the Bologna (SISMER) center for poor- prognosis patients undergoing IVF/ICSI. A steady increase of the referral of IVF patients for PGD was observed during the last two years, with almost of one third performed during the last year (Gianaroli and collaborators, unpublished data).
Overall, the above four active centers, including Chicago, St. Barnabas, Brussels and Bologna, performed 2774 PGD cycles, resulting in 2265 transfers and 652 clinical pregnancies (29%). More than one quarter of these cycles have been offered since the last meeting, with the majority of PGD cycles still being done for the age-related aneuploidies, which in contrast to other indications are mainly applicable to poor-prognosis patients, including those of advanced age.
Because of the demonstrated clinical significance of PGD for both prenatal genetic care and assisted reproduction, PGD is presently applied not only in the Western Europe and USA, but also in the Eastern Mediterranean, Southeast Asia and Western Pacific regions. For example, PGD was applied in Cyprus and Greece as part of a programme for the prevention of hemoglobin disorders (Kuliev et al. 1999; Kanavakis et al. 1999). In these two programmes, approximately 80 PGD cycles were performed for thalassemia and sickle cell disease, resulting in at least 30 clinical pregnancies and the birth of a dozen healthy children. In Turkey, PGD was implemented as part of assisted reproduction, and applied for aneuploidy testing in 134 IVF cycles for advanced maternal age, repeated implantation failure or abnormal gamete cell morphology. Pronuclear morphology in this series was also scored in 51 cycles, and shown to be a possible indicator of a poor outcome following the transfer of aneuploidyfree embryos. Of 129 transfer cycles, 47 pregnancies were achieved (36.4%), resulting in birth of 42 healthy babies, with 10 pregnancies still ongoing. All four pregnancies resulting in spontaneous abortions were in the abnormal gamete cell morphology group (Kahraman and collaborators; Bahce and collaborators; both unpublished data).
A total of 32 PGD cycles were performed for younger infertile patients undergoing ICSI in Jordan, using aneuploidy testing of polar bodies or blastomeres. Embryos were transferred in 20 cycles where at least one aneuploidy-free embryo was detected, yielding five clinical pregnancies and six normal babies (Haj Hassan and collaborators, unpublished data).
PGD for variety of indications was reported from the Sidney center including aneuploidies, translocations and single gene disorders. This included PGD for mitochondrial mutations, which was attempted for the first time. It is of interest, that a single biopsied blastomere in this case was found to be representative to detect the mutation load of the corresponding embryo, suggesting that it should be possible to screen for oocytes or embryos with lower mutation levels and which may give higher pregnancy rates after transfer (Leigh and collaborators, unpublished data).
As seen from the overall analysis of the present PGD experience, the most universal PGD application was the testing for aneuploidies in poor prognosis IVF patients. This experience presently exceeds 2000 cases, and has yielded more than 500 clinical pregnancies and approximately 470 healthy newborn babies. It is highly possible that aneuploidy testing may soon become a required standard of care for assisted reproduction, especially for IVF patients of advanced maternal age. This approach would replace the present practice of “blind” embryo selection on morphological grounds.
Analyses of experience of more than 3000 PGD cycles performed until today further demonstrate the clinical value of pre- pregnancy testing for preventing genetic disorders and improving the standards of assisted reproduction. An increasing number of centers have presently each performed more than hundred PGD cycles, illustrating the safety, accuracy and reliability of PGD for genetic and chromosomal disorders. As mentioned earlier, only four of the above mentioned active centers have accumulated an experience comprising more than three quarters of the overall PGD cycles. Their data on outcomes of almost 700 clinical pregnancies is much larger than those available from the multi-center collection (ESHRE Preimplantation Genetic Diagnosis Consortium, 2000). For example, results of more than 2,000 cycles performed for aneuploidies suggest the practical relevance of aneuploidy testing as part of the future of overall IVF practices. They also indicate a need for the provision of adequate services for infertile couples of advanced maternal age.
It is also obvious that to be appropriate, current genetic counseling services have to inform patients at high genetic risk about the availability of PGD. Without this information, couples objecting to traditional prenatal diagnosis have to remain childless because of their fear of pregnancy termination. Many couples will also have to control their reproduction after one or two pregnancy terminations following prenatal diagnosis. Some couples can carry translocations resulting in up to 90% risk of spontaneous abortions. Data presented here suggest as much as a nine-fold reduction of spontaneous abortions that could occur in these patients following PGD. Awareness of the availability of PGD will permit such couples to establish pregnancies unaffected from the onset and have children of their own.
PGD has also provided a unique opportunity for couples at risk for the late onset disorders with genetic predisposition. They may now plan on having progeny free from genetic predisposition, without facing a risk for termination of pregnancy. A strong predisposition to late onset disorders can be detected by prenatal diagnosis, yet this has never been an indication for traditional prenatal diagnosis because of a potential pregnancy termination, which cannot be justified by a genetic predisposition alone. PGD, on the other hand, has made it realistic to establish only pregnancies completely free of such genetic predisposition, thus making pre-pregnancy diagnosis for such conditions perfectly acceptable on ethical grounds. This benefit has recently been realized clinically through PGD for cancer predisposition, resulting in the birth healthy child free from Li-Fraumeni syndrome, which is a familial predisposition to breast cancer, bone and soft tissue sarcomas and brain tumor, determined by p53 tumor-suppressor mutation (Verlinsky et al. 2001).
Finally, PGD has opened a prospect for a non-disease testing, which provides opportunities to combine pre-selection of mutation-free embryos with HLA matching. At-risk couples may establish pregnancy with an unaffected child, to provide HLA matched progeny for treatment of an existing affected sibling utilizing bone marrow transplantation from the family member. On the other hand, the future application of this approach for non-disease testing as a primary indication will need careful ethical consideration.
The next annual meeting will be held in association with 4th International Symposium on Preimplantation Genetics in Limassol, Cyprus, April 11-13, 2002.
Y. Verlinsky, L. Gianaroli, A. Kuliev, P. Braude, I. Liebaers, R. Edwards, T. Escudero, S. Rechitsky, D. Wells, D. Cram, D. Leigh, R. Martin, C. Ogilvie, A. Lashwood, M. Monk, C. Magli, S. Kahraman, M. Bahce, K. Yakin, L. Haj Hassan, J. Wolstenholm, H. Clouston, G. Kalakoutis, G. Tsukerman, C. Hanson, S. Kolvraa, J. Santolaya-Forgas, Y. Wettergren, M. Petrou, B. Heck, I. Kirillova, C. Canton, S. Zou, L. Govaerts, E. Elsobky, L. Gadea.
1. International Working Group on Preimplantation Genetics. 1999 Preimplantation Diagnosis: An Alternative to Prenatal Diagnosis of Genetic and Chromosomal Disorders. Report of the 8th Annual Meeting International Working Group on Preimplantation Genetics, in association with the 9th International Conference on Prenatal Diagnosis and Therapy, Los Angeles, June 7, 1998. Journal of Assisted Reproduction and Genetics, 16, 161-164
2. International Working Group on Preimplantation Genetics. 2000 Preimplantation Diagnosis: An Integral Part of Assisted reproduction. 2000 Report of the 9th Annual Meeting International Working Group on Preimplantation Genetics, in association with the 11th IVF Congress, Sydney, May 10, 1999. Journal of Assisted Reproduction and Genetics, 17, 75-79
3. International Working Group on Preimplantation Genetics. 2001 10th Anniversary of Preimplantation Genetic Diagnosis. Report of the 10th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 3rd International Symposium on Preimplantation Genetics, Bologna, June 23, 2000. Journal of Assisted Reproduction and Genetics, 18, 66-72.
4. Verlinsky Y, Evsikov S. 1999 Karyotyping of human oocytes by chromosomal analysis of the second polar body. Molecular Human Reproduction, 5, 89-95.
5. Evsikov S, Verlinsky Y 1999 Visualization of chromosomes of single human blastomeres. Journal of Assisted Reproduction and Genetics 16,133-137.
6. Willadsen S, Levron J, Munne S, et al. 1999 Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Human Reproduction, 14, 470-475
7. Munne S, Morrison L, Fung J, et al. 1998 Spontaneous abortions are significantly reduced after preconception genetic diagnosis of translocations. Journal of Assisted Reproduction and Genetics, 15, 290-296.
8. Evsikov S, Cieslak J, Verlinsky Y. 2000 Survival of unbalanced translocations to blastocyst stage. Fertility and Sterility, 74, 672-676.
9. Wells D, Delhanty DA. 2000 Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Molecular Human Reproduction, 6, 1055-1062.
10. Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blazoners from human embryo by using comparative genomic hybridization. Human Genetics, 106, 210-217.
11. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. 2001 Preimplantation diagnosis for Fanconi anemia combined with HLA matching. Journal of the American Medical Association, 285, 3130-3133
12. Rechitsky S, Verlinsky O, Strom C, et al. 2000 Experience with single-cell PCR in preimplantation genetic diagnosis: how to avoid pitfalls. In: Hahn S, Holzgreve W. (eds); Fetal Cells in Maternal Blood, pp 8-15. New Developments for a New Millennium. 11th Fetal Cell Workshop, Basel. Karger, Basel.
13. ESHRE Preimplantation Genetic Diagnosis Consortium. 2000 Data Collection II, May 2000. Human Reproduction, 15, 2673-2683.
14. Verlinsky Y, Rechitsky S, Verlinsky O, et al. 2001 Preimplantation diagnosis for p53 tumor suppressor gene mutations. Reproductive BioMedicine Online, 2, 102-105.
15. Munne S, Magli C, Cohen J, et al. 1999 Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Human Reproduction, 14, 2191-2199.
16. Kuliev A, Rechitsky S, Verlinsky O, et al. 1999 Birth of healthy children after preimplantation diagnosis for thalassemias. Journal of Assisted Reproduction and Genetics, 16, 185-189.
17. Kanavakis E, Vrettou C, Palmer G et al. 1999 Preimplantation genetic diagnosis in 10 couples at risk of transmitting beta-thalassaemia major: clinical experience, including initiation of six singleton pregnancies. Prenatal Diagnosis, 19, 1217-1222.
18. Edwards RG. 2000 A well-taken opportunity for a double blessing. Reproductive BioMecine Online 1, 31-33.
19. Damewood MD 2001 Ethical implications of a new application of preimplantation diagnosis. Journal of the American Medical Association, 285, 3143-3144