Molecular Human Reproduction, Vol. 9, No. 5, 301-307,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
An evaluation of PGD in clinical genetic services through 3 years application for prevention of ß-thalassaemia major and sickle cell thalassaemia
Submitted on October 17, 2002; accepted on February 8, 2003
1 Medical Genetics, Athens University and 2 Athens University Research Institute for Prevention and Treatment of Genetic and Malignant Diseases of Childhood, St Sophia’s Children’s Hospital, Athens 11527 and 3 Embryogenesis, Centre for Reproductive and Fertility Studies, Athens 15125, Greece 4 Present address: IVF Clinic, Mitera Maternity Hospital, Athens 15123, Greece
5 To whom correspondence should be addressed. e-mail: ekanavak{at}cc.uoa.gr
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ABSTRACT |
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PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of ß-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.
Key words: ß-thalassaemia major and HbS/genetic services/PGD/prenatal diagnosis
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Introduction |
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PGD represents an alternative procedure in the context of prenatal diagnostic services. The advantage over conventional prenatal diagnosis is the avoidance of terminating affected pregnancies. It involves identification and selective transfer of unaffected IVF embryos in couples at risk of transmitting a genetic disorder, and thus requires combined expertise in the fields of reproductive medicine and genetics (Edwards and Hollands, 1988; Handyside et al., 1989). PGD procedures most commonly involve blastomere biopsy from 3-day cleavage stage embryos, and for monogenic disorders, subsequent diagnostic protocols based on the PCR (Harper and Delhanty, 2000; Kanavakis and Traeger-Synodinos, 2002). Although the first PGD cycles were performed more than a decade ago (Handyside et al., 1989), uptake in the framework of genetic services has been slow. This is probably due to the need to involve expertise in both reproductive medicine and genetics, the fact that all stages of the multi-step procedure are technically challenging, and also because the transfer of genetic diagnostic protocols to the single cell level has proved surprisingly difficult (Harper and Delhanty, 2000).
The thalassaemia syndromes and related haemoglobinopathies are the commonest group of monogenic disorders world-wide (Weatherall, 2000). ß-thalassaemia is an autosomal recessive disorder caused by mutations in the ß-globin gene located on chromosome 11. More than 170 point mutations or small insertions/deletions have been described which either reduce or abolish the synthesis of the ß-globin chains of adult haemoglobin (HbA) by the affected gene (http://globin.cse.psu.edu/globin). People who inherit two ß-thalassaemia mutations usually have ß-thalassaemia major, a severe dyserythropoietic anaemia requiring life-long treatment with blood transfusions to maintain satisfactory levels of haemoglobin and iron-chelation therapy to combat the tendency to iron overload. In many countries throughout the world carrier detection programmes, with the option of prenatal diagnosis, are well established, in order to reduce the estimated 70 000 affected new births expected annually (Cao et al., 1998). The sickle cell syndromes are also caused by mutations in the ß-globin gene, and homozygotes or compound heterozygotes are characterized by life-long haemolytic anaemia with increased morbidity and mortality associated with a variety of complications related to the deleterious effects of vaso-occlusive episodes and increased propensity to infection. In Greece almost 10% of the population are carriers of ß-thalassaemia or HbS, and 
600 couples are expected to request prenatal diagnosis annually (Loukopoulos, 1996; Kanavakis et al., 1997).
To integrate the option of PGD within our prenatal diagnosis programme for ß-thalassaemic haemoglobinopathies in Greece, for couples with an unsuccessful or problematic reproductive history, we established an accurate, reliable single-cell genotyping protocol capable of diagnosing the wide spectrum of ß-globin gene mutations found in Greece. This involved pre-clinical experiments on 490 single cells (blastomeres from supernumary human embryos, lymphocytes and amniocytes) in order to optimize accuracy of the genotyping method, whilst simultaneously addressing inherent problems of single-cell PCR, including PCR failure, allele drop-out and contamination (Kanavakis et al., 1999; Vrettou et al., 1999). The optimized genotyping method achieved a PCR efficiency of 85–90% and <8% allele drop-out, and is based on the mutation scanning method, denaturing gradient gel electrophoresis (DGGE) (Vrettou et al., 1999).
In this report of more than 3 years experience with clinical PGD cycles we present many of the positive and negative aspects of PGD when applied in the context of a preventive genetic service for a common recessive disease (the ß-haemoglobinopathies), highlighting approaches for improvement, pitfalls and overall limitations.
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Materials and methods |
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Subjects
Over a 3 year period, 59 couples at risk for transmitting homozygous ß-thalassaemia or sickle cell thalassaemia requested counselling for PGD. All couples underwent an initial consultation at both the IVF unit and genetics department to provide information on all stages of the IVF/PGD procedure, the success rates for achieving an unaffected live birth, and to discuss the reason(s) for requesting PGD (ESHRE PGD Consortium, 2002). All couples were advised to undergo conventional prenatal diagnosis for any pregnancy established, since the PGD genotyping method we intended to use had not been previously applied in a clinical context. During the initial consultation at the genetics department, blood samples (in EDTA) were collected for haematological and DNA analyses (Kanavakis et al., 1997). Those couples proceeding with a PGD cycle were advised to contact the IVF unit to programme cycle initiation. All patients in the study gave written informed consent.
Patient treatment and embryo culture
In 41 couples who have so far initiated PGD cycles (total 63 cycles), female patients had hormonal treatment for follicle stimulation, and oocyte retrieval was performed 14 days later according to standard IVF procedures (Kanavakis et al., 1999; ESHRE PGD Consortium, 2002). Prior to ICSI, the oocytes were stripped of cells, to eliminate contamination by maternal cells when performing embryo biopsy. The ICSI protocol was perfomed as previously described (Van Steirteghem et al., 1993), and is recommended for PGD to prevent contamination by spermatozoa when performing embryo biopsy (ESHRE PGD Consortium, 2002; Kanavakis and Traeger-Synodinos, 2002). Oocytes exhibiting two pronuclei 16 h post-insemination (p.i.) were considered fertilized (Kanavakis et al., 1999; Palmer et al., 2002).
Biopsy procedure and embryo transfer
Blastomere biopsies were performed on the third day p.i. when the embryos had reached five to eight cells. Biopsies were undertaken using the acid Tyrode’s protocol (ZD-10; Scandanavian IVF Sciences, AB, Sweden) for zona drilling (Hardy et al., 1990). A single blastomere was biopsied from each embryo and placed in an 0.2 ml Eppendorf tube containing 10 µl of double-distilled sterile water, overlaid with mineral oil (all DNAse and RNAse free), and placed at –20°C. Immediately following biopsy, embryos were transferred to separate wells of Nunclon dishes containing culture medium (S2 or G2.2; Scandanavian IVF Sciences, AB, Sweden), until embryo transfer on days 4 or 5 p.i. following genotype analysis. Embryos diagnosed as unaffected for ß-thalassaemia major or sickle cell thalassaemia were transferred using standard procedures (Kanavakis et al., 1999; Palmer et al., 2002).
Cell lysis and genotype analysis
Early on day 4 p.i., the biopsied blastomeres were removed from the freezer, proteinase K (Roche Diagnostics GmbH, Manheim, Germany) was added to each tube under stringent conditions (to a final concentration of 50 µg/ml) and the biopsied blastomeres were transfered in their sealed tubes from the IVF unit to the genetics laboratory for analysis. Immediately on receipt at the genetics laboratory, the blastomeres were lysed by proteinase K treatment (37°C for 1 h and 65°C for 10 min), followed by inactivation of the enzyme at 95°C for 10 min (Vrettou et al., 1999).
Genotype analysis involved a first round of PCR to amplify one of two alternative regions of the ß-globin gene according to the parental genotypes (Figure 1 and Table I), followed by nested-PCRs within the first-round region to produce DNA fragments suitable for analysis using DGGE; sequences of all PCR primers were as previously described (Palmer et al., 2002). Following the nested-PCR, amplification products were checked by agarose gel electrophoresis and PCR positive samples were analysed by DGGE as previously described (Kanavakis et al., 1997; Palmer et al., 2002) (Figure 2).
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Stringent precautions against contamination were applied for all PGD cycles performed, involving PCR set-up in an isolated UV-treated area, the use of exclusive equipment and stringently prepared reagents. Pre- and post-PCR procedures were strictly separated and each PGD analysis was monitored by the inclusion of several negative controls and blanks at all stages (Vrettou et al., 1999; Palmer et al., 2002).
Early day 5 p.i., the genotype of each blastomere was evaluated from the DGGE gel, the results were reported to the IVF unit and unaffected embryos were transferred to the patient.
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Results |
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All 59 couples were considered suitable for PGD based on their reason(s) for requesting PGD as opposed to conventional prenatal diagnosis (Table II). Forty-one were subfertile (Table II), 12 of whom had also selectively terminated an IVF pregnancy following an affected prenatal diagnosis. Of 13 couples without fertility problems but who wished to avoid termination of pregnancy (TOP), 10 had experienced selective abortion for at least one normally conceived pregnancy, and three had a child affected with ß-thalassaemia major. In five couples one partner had ß-thalassaemia major and the other was a ß-thalassaemia (or
ß-thalassaemia) carrier, thus increasing the risk of conceiving an affected pregnancy to 50% (Table II); one of these couples had already selectively terminated two naturally conceived affected pregnancies detected by chronic villous sampling (CVS).
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Based on the ß-globin gene mutation interactions, 57 couples were advised that PGD analysis was technically feasible with the established PCR/DGGE protocol (Vrettou et al., 1999) (Figure 1 and Table I). There were 30 genotype combinations, involving the diagnosis of 11 different ß-thalassaemia mutations and HbS. Diagnosis of genotypes involving
ß-thalassaemia alleles (ß-Lepore and ß-Sicilian) could be achieved indirectly through the use of linked polymorphisms (Figure 1 and Table I), and also by exclusion through detection of a normal allele. However, the mutation interaction involving HbS with HbD (occurring in two couples) could not be diagnosed with the present nested-PCR/DGGE protocol; the causative mutation for HbD lies outside the region of the ß-globin gene analysed with this method, and neither couple had informative linked polymorphisms (Figure 1) (Vrettou et al., 1999).
In 17 couples, the potential affected genotype included a milder ß-thalassaemia mutation (IVSI-nt6T>C) or ß-thalassaemia (Table I), which may cause thalassaemia intermedia rather than thalassaemia major. Although this question was raised during counselling, no couple was willing to risk the outcome of a pregnancy without PGD and/or prenatal diagnosis, as there are no definite genetic predictors for disease severity (Ho et al., 1998).
Forty-one couples initiated at least one PGD cycle (Table II). Ten couples are waiting to start a PGD cycle, including two couples with HbS plus HbD for whom we are developing an appropriate genotyping method (Table I). Five couples have been lost to follow-up after the initial counselling and DNA analysis, and three have reported initiation of a naturally conceived pregnancy.
The 63 PGD cycles included one cycle in 22 couples, two cycles in 16 couples and three cycles in three couples (Tables I and II). Twenty cycles were cancelled before the biopsy stage, either due to low numbers of oocytes collected (<8), and/or poor quality embryos (Table II and Figure 3). Following counselling, six subfertile couples (six cycles) with low numbers of embryos chose to have embryos transferred without PGD, so as not to waste the first stages of an IVF cycle (data not shown). Amongst 32 couples completing at least one PGD cycle, genotype analysis involved detection of one ß-thalassaemia mutation in nine couples, and potentially compound mutation interactions in 23 couples (including two couples with a ß-thalassaemia major patient and a ß-thalassaemia heterozygote partner, necessitating detection of three potential mutations within each blastomere) (Table I). No contamination of PCR reagents or biopsy media was evident in any of the cycles, as indicated by the negative controls and blanks at all stages.
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The outcome at each stage of the PGD cycles is summarized in Figure 3, and overall led to the transfer of at least one unaffected embryo in the 43 cycles for which genetic analysis was performed. In the first six cycles, embryos were transferred on day 4 p.i. and in 37 cycles embryos were transferred on day 5 p.i., by which time 40% had developed to blastocysts. Thus, unaffected mixed blastocysts and non-blastocysts were transferred in 31 cycles, and unaffected day 5 non-blastocysts in six cycles (Palmer et al., 2002). Following embryo transfer, 16 women had positive HCG levels (37% pregnancy rate for cycles completed and 25% for all cycles initiated), 13 of which were confirmed with fetal sacs and heart beat, including 10 singletons, two twins and one triplet pregnancy (17% implantation rate per embryo transferred) (Figure 3).
Two singleton pregnancies spontaneously miscarried within the first trimester, and one singleton pregnancy was ectopic and had to be terminated. Prenatal diagnosis was performed in the remaining 10 pregnancies using either CVS sampling (six cases) or amniocentesis (four cases) (Table III). All babies in the triplet and two twin pregnancies were confirmed as unaffected for ß-thalassaemia major (or ß/ß-thalassaemia), although the triplet pregnancy was (successfully) reduced to twins at the request of the parents. Of the six singleton pregnancies, five were confirmed as unaffected, but one was detected as a PGD misdiagnosis and was selectively terminated at 12 weeks. The nine unaffected pregnancies all went to term without complications, and have resulted in the birth of 12 full-term healthy babies (Table III). All 12 babies, delivered by Caesarian section (without specific indication but at the discretion of the gynaecologists) after 36–37 weeks, were of satisfactory weight, with no apparent abnormalities. The present ages of the babies range from several months to >2 years. All are reported to have had a normal neonatal period (and early childhood).
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Discussion |
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The main approach for controlling severe genetic diseases remains prevention, and PGD represents an alternative procedure in the context of prenatal diagnostic services, heralded as a means to avoid the trauma and ethical dilemma of terminating affected pregnancies. Although it represents a procedure which potentially incorporates some of the latest advances in biomedical technology, clinical application has been relatively limited. We approached the problem of offering PGD within the context of prenatal diagnostic services for a common genetic disorder in Greece, and our experience over more than 3 years highlights many of the positive aspects and some of the potential pitfalls and limitations.
A positive outcome of a PGD cycle (i.e. an unaffected full-term baby), depends upon the success of the assisted reproductive treatment, as well as the genetic diagnosis. It is only the stage of genetic diagnosis that can be more stringently standardized, whereas the outcome of each stage of assisted reproductive treatment tends to be case dependant.
Let us discuss the stage of genetic diagnosis. When confronting a common and heterogeneous monogenic disease such as the ß-haemoglobinopathies, it is more practical to have a single PGD diagnostic strategy applicable for a wide spectrum of potential affected genotypes, rather than designing and standardizing case-specific protocols each time. Several methods applicable to PGD of haemoglobinopathies have been described for detection of some of the numerous ß-gene mutations (Holding and Monk, 1989; Ray et al., 1996; Kuliev et al., 1998; De Rycke et al., 2001), but only a few have been described for potentially wider application (El-Hashemite et al., 1997; Vrettou et al., 1999; Piyamongkol et al., 2001). Besides successfully defining the disease-associated genotype, any protocol must also address many of the inherent problems potentially associated with PCR-based genotyping of single cells (PCR failure, allele drop-out and contamination), to ensure that the method is reliable and accurate for clinical application (Wells and Sherlock, 1998). All methods have to be worked up on single cells with known genotypes and laboratories tend to select methods with which they already have experience. The method used in this study, which applies a nested-PCR protocol producing amplicons analysable with DGGE, was accurate in trials and optimized to give <10% PCR failure and allele drop-out (ADO) (Vrettou et al., 1999). DGGE is a very reliable method for genetic diagnosis once a DGGE pattern detected in a sample has been associated with a specific mutation (Losekoot et al., 1990). We felt that it would be particularly advantageous for single-cell genotyping, since as a scanning method it can identify many mutations in a single amplified region, avoiding both the need for independent mutation assays, and facilitating simultaneous analysis of more than one mutation in a single PCR fragment (for compound genotypes).
In this relatively small series there were 30 mutation interactions, 29 of which could be diagnosed by this method (Figure 1 and Table I). DGGE is also appropriate for PGD since it monitors the occurrence of ADO when ‘impossible’ genotypes are obtained. This not only applies when analysing single DGGE fragments, but also when analysing compound genotypes with mutations in neighbouring fragments, assuming that they have been amplified from the same first-round PCR product. Most importantly, DGGE is an assay for the presence of normal as well as pathological alleles, such that only blastomeres with definitive evidence of a normal allele on DGGE analysis are considered unaffected, preventing transfer of affected embryos even if ADO has occurred (Vrettou et al., 1999). Analogous methods based on SSCP analysis have been described for PGD of ß-thalassaemia, although their clinical application has been limited (El-Hashemite et al., 1997; Piyamongkol et al., 2001).
In this series, the genotyping method was reliable, with a PCR success rate (and thus diagnosis) in 78% of blastomeres analysed. ADO varied widely between cycles, ranging from no apparent ADO in 13 cycles to as high as 60% in two cycles (data not shown). Although the cause(s) of ADO remain generally unknown (Kanavakis and Traeger-Synodinos, 2002), its occurrence may be influenced by some of the factors that affect PCR efficiency. First, the often sub-optimal quality of genetic material reported for many blastomeres from IVF embryos may influence both PCR success and occurrence of ADO, but this factor is obviously case dependant (Wells and Delhanty, 2000). However, the slightly lower PCR success rate in PGD cycles with our method could be partly attributed to the relatively large first-round PCR product, possibly reducing single-cell PCR efficiency (Sermon et al., 1996; Vrettou et al., 1999), and also to the lower detection sensitivity of non-fluorescently labelled PCR amplicons. With respect to the former, we feel that the wide applicability of the method outweighs the slightly lower PCR efficiency, but we are presently developing an analogous genotyping method based on real-time fluorescent PCR in order to improve sensitivity of amplicon detection.
The accuracy of the genotyping method was previously established in trials (Vrettou et al., 1999), and has been monitored through re-analysis of 53 non-transferred embryos in completed cycles, which were all in accordance with the PGD results. However, we disappointingly had a single case of misdiagnosis detected amongst 14 babies (10 pregnancies) evaluated by prenatal diagnosis. The case in question occurred in a subfertile couple who carried different ß-thalassaemia point mutations located in the same DGGE analysable fragment. On the basis that all embryos designated as unaffected have a normal allele with DGGE analysis (Figure 2), we tend to conclude that the misdiagnosis was not due to inaccuracy of the genotyping method but rather to chance contamination of a tube(s) of the cycle in question by extraneous genetic material introduced during processing (Lewis et al., 2001), or a tube-switch, reflecting limitations of the present technology, which involves ‘hands-on’ multi-step protocols. Several PGD protocols have now been described which monitor chance extraneous contamination of individual tube(s), through the inclusion of a polymorphic marker multiplexed within the genotyping assay (Piyamongkol et al., 2001; Harper et al., 2002) and we are investigating the inclusion of at least one such marker known to have a high rate of heterozygosity in the Greek population (Traeger-Synodinos et al., 1998).
The discordance between frequency of unaffected embryos diagnosed (52%) versus expected (near 75%), implies that about half of embryos rejected for transfer may be unaffected. This discrepancy is consistent with other reports of PGD for recessive monogenic diseases (ESHRE PGD Consortium, 2002), and may be attributed to a small degree to ADO, but also to the poor genetic quality of many IVF embryos (Wells and Delhanty, 2000). The latter is a potentially limiting factor when performing PGD generally, although it should not lead to transfer of an affected embryo for a monogenic recessive disease.
Let us turn now to the aspects of assisted reproduction in PGD. A major obstacle to a wider application of PGD is the necessary involvement of assisted reproduction tehcniques (ART), even if the couple do not have fertility problems. The majority of couples in this series had some form of subfertility (75%) and planned to undergo IVF to establish a pregnancy anyway (Table II), although this bias was because most referrals were through the collaborating IVF unit. The ESHRE PGD Consortium reports that generally about a quarter of all couples requesting PGD (for either chromosomal or monogenic disorders) need ART which they wish to combine with PGD. However, genetic risk with the wish to avoid pregnancy termination, usually following experience of an affected pregnancy or birth of an affected child, is usually the most common reason for requesting PGD (ESHRE PGD Consortium, 2002). In addition, PGD is a relevant option for ß-thalassaemia major female patients with heterozygote spouses, who have a 50% risk of an affected pregnancy. With improved treatment of thalassaemia major, more patients attain adulthood and reproductive capacity, and such patients may not fair well with repeated selected terminations following a first or second trimester prenatal diagnosis (Skordis et al., 1998; Perniola et al., 2000).
The outcome of IVF/ICSI treatment and the quality of embryos available for biopsy are overall case dependent, and are influenced by whether the couple is fertile, the underlying cause of subfertility and the age of the woman (Edwards and Beard, 1999; Van de Velde et al., 2000), although the consequence of each of these factors cannot be evaluated in this small series. About one third of cycles initiated in this study were cancelled before embryo biopsy and genetic analysis, with nine of 41 couples failing to complete even a single PGD cycle, despite several attempts by some couples. About one third of completed PGD cycles resulted in pregnancy initiation, although there was a relatively high rate of early pregnancy loss, whereby only two thirds of pregnancies reached the second trimester, without apparent explanation (European IVF-Monitoring Programme, 1997; ESHRE PGD Consortium, 2002). Overall less than one quarter of couples who initiated a PGD cycle achieved an unaffected baby. Surprisingly, although the majority of couples in our cohort had some form of subfertility, these rates of pregnancy and babies born are comparable to those reported to the ESHRE PGD consortium overall, and which do not differ notably from those for IVF generally (European IVF-Monitoring Programme, 1997). Until the outcome of ART in PGD has been more fully evaluated, couples should be carefully counselled when they request PGD. For subfertile couples requiring IVF to assist pregnancy initiation, the selection of unaffected embryos by PGD potentially reduces the number of embryos available for transfer (although any pregnancy initiated will be unaffected). On the other hand, couples requesting PGD who do not have fertility problems or experience of a previous affected pregnancy, should weigh up the stress of IVF and the relatively low PGD pregnancy rates against the high likelihood of naturally conceiving an unaffected pregnancy, but with the stress of undergoing prenatal diagnosis, and possible pregnancy termination.
The tactic that we follow of biopsing on day 3, but transferring on day 5, by which time many embryos have developed to the blastocyst stage, does not appear to be detrimental for establishing pregnancies, and we prefer this strategy to increase the time available for the genetic diagnosis (Gardner et al., 1998; Edwards and Beard, 1999; Palmer et al., 2002). Although many PGD centres recommend the biopsy and replicate analysis of two blastomeres from each cleavage stage embryo (Van de Velde et al., 2000; ESHRE PGD Consortium, 2002), we found that this approach was limited practically, as only a minority of embryos reached seven to eight cells on day 3 p.i. Thus, we selected to biopsy one blastomere in all embryos.
Relative to the safety of PGD, the babies in this series were all apparently healthy, consistent with other reports on PGD babies (Strom et al., 2000; ESHRE PGD Consortium, 2002), although more data is required before safety can be fully assessed.
From a totally practical viewpoint, PGD is a multi-step, labour intensive procedure, requiring highly experienced operators for both manipulation of embryos and the genetic analysis. PGD cycles have to be well planned in order to avoid compromising other working commitments of the team(s) involved, since biopsy and diagnosis have to be performed within a very specific and limited time margin. In addition, the relatively high cost of the IVF/PGD procedure may also prevent wider uptake.
Overall our experience of clinical PGD cycles integrated as part of a prevention programme for a common monogenic disorder highlights that, with present medical expertise, the stages of ART may be a limiting factor for a positive outcome of a PGD cycle (i.e. birth of an unaffected baby). Protocols for the genetic analysis can be rigorously standardized, but with present ‘hands-on’ technology may be subject to chance errors. With incorporation of new technologies, accumulating experience and continuing research efforts, PGD should play an increasing role as a specialized clinical procedure, for it has proved highly worthwhile for many couples with a problematic reproductive history and a high risk of transmitting a genetic disease (ESHRE PGD Consortium, 2002). However, in the context of preventive genetic services PGD is unlikely to completely replace prenatal diagnosis.
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Acknowledgements |
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The authors wish to thank Stavroula Papadopoulou for technical assistance. This work was partly funded by an Athens University Research Grant (ELKE no. 70/4/4255). Christina Vrettou is supported by a post-doctoral scholarship from the Greek State Scholarship Foundation.
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