1 Unità di Medicina della Riproduzione, Associazione HERA, Catania, and 2 Servizio di Talassemia, Ospedale V. Cervello, Palermo, Italy
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Abstract |
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Key words: allele drop-out/ß-thalassaemia/clinical application/preimplantation genetic diagnosis/sickle cell anaemia
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Introduction |
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Nowadays, PGD is widely applied using fluorescence in-situ hybridization (FISH) for chromosomal disorders, or PCR for single-gene disorders. The ESHRE PGD Consortium Steering Committee reported data on 1318 PGD cycles, 163 pregnancies and 162 babies (ESHRE PGD Consortium Steering Committee, 2000). In the present study, PGD was applied to couples that risked transmitting ß-thalassaemia and sickle cell anaemia, and embryos were transferred with wild-type genotype or heterozygous for one wild-type allele.
The Sicilian population runs a high risk of transmitting the autosomal recessive diseases ß-thalassaemia and sickle cell anaemia. The average gene frequency for these two diseases are 6 and 1% respectively, with ~350 000 carriers. It has been estimated that 1 in 270 couples risks transmitting these ß-globin gene disorders, with 66 new births per year (Caronia et al., 1989; Giambona, 1995
). Before starting the present programme of PGD for ß-thalassaemia and sickle cell anaemia, a survey was carried out to test the willingness of high-risk Sicilian couples to undergo PGD. It was found that 44.4% of couples attending for their first PND, 47.1% of couples attending for their second or further PND without previous experience of therapeutic abortion, and 72.0% of couples undergoing PND with previous experience of therapeutic abortion were willing to undergo PGD for ß-thalassaemia and sickle cell anaemia (Chamayou et al., 1998
). In addition, an investigation was carried out to determine which cellular lysis protocol provides the greater amplification efficacy and lesser allele drop-out (ADO) on single blastomeres obtained from surplus embryos that are carriers of one ß-globin allele mutation. In the present study, nine cycles of clinical PGD application for ß-thalassaemia and sickle cell anaemia are reported, in two fertile carrier couples with previous experiences of therapeutic abortion for an affected fetus and five infertile carrier couples.
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Materials and methods |
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Both partners of couples A, B and E had the same ß-globin mutated allele (Cd39). Both partners of couples C, D, F and G were heterozygous for two different ß-globin mutated alleles. Couples A and C underwent two PGD treatments (noted A' and C'). The average age of the women patients undergoing PGD for ß-thalassaemia and sickle cell anaemia was 31.4 years.
ICSI and embryo culture
In the IVF programme, controlled ovarian stimulation was carried out for all patients by the administration of GnRH analogue (Suprefact®; Hoechst Marion Roussel Deutschland GmbH, Germany) in a long protocol, followed by recombinant FSH (Gonal-F®; Ares-Serono Ltd, England or Puregon®; Organon N.V. Organon, Holland) from cycle day 3. Ovulation was induced by HCG 10 000 IU (Profasi®; Ares-Serono). Oocyte retrieval was carried out using vaginal ultrasound-guided aspiration, and the oocytes were cultured in IVF Universal medium (Medicult, Denmark) and incubated at 37°C in 5% CO2 in air. The oocytes were micro-injected with the partner's sperm using standard ICSI on the same day (day 0) (Palermo et al., 1992; Van Steirteghem et al., 1993
). On day 1, normal fertilization was carefully checked by the observation of pronuclei. The in-vitro culture was prolonged in IVF Universal medium until the 4-cell embryo stage, and continued further in M3 medium (Medicult, Denmark). The biopsy was performed at the 6-to 8-cell embryo stage (morning of day 3). After embryo-biopsy, in-vitro culture was continued in M3 medium until the result of the genetic diagnosis for ß-thalassaemia and/or sickle cell anaemia had been obtained. Embryo transfer was carried out on the morning of day 4, using those embryos that continued to show good in-vitro development.
Biopsy procedure
The method used has been described previously (Hardy et al., 1990; Tarin and Handyside, 1993
). The biopsies were performed using an inverted microscope (Olympus IX70, Japan) equipped with Hoffman optics and Narishige manipulators (Japan). Two Narishige MMO-202D manipulators and two Narishige MM-88 micro-manipulators controlled three pipettes, to each of which was attached micro-injectors. The embryo was immobilized using a 120 µm outer-diameter holding micro-pipette in a 5 µl drop of M3 medium buffered with 25 mmol/l HEPES, and under mineral oil. A minimal quantity of Tyrode's acid solution was delivered by drilling a hole in the zona pellucida with a 10 µm inner diameter micro-pipette. A 35 µm inner diameter micro-pipette was introduced through the opening and one blastomere was aspirated out of the zona pellucida. After the biopsy procedure, each embryo was washed three times in M3 medium and incubated in 20 µl of M3 medium. The biopsied blastomere was washed three times in sterile phosphate-buffered saline (PBS) solution and transferred into a 0.2 ml Eppendorf tube containing 2 µl of sterile PBS solution. Its presence was carefully checked in the Eppendorf tube under a Nikon SMZ-2T stereo-microscope (Japan). Each blastomere sample was immediately conserved at 20°C until the biopsy procedure had been completed for all available embryos. If the cytoplasmic membrane of the first biopsied blastomere was opened during the biopsy procedure, a second blastomere was removed and conserved. This second blastomere was sampled into the same Eppendorf tube as the preceding one. If the embryo had seven or more cells, a second blastomere was biopsied and transferred into a second Eppendorf tube with a view to comparing the genetic diagnosis of two cells from the same embryo.
Cell lysis
Having previously determined that a minor ADO rate was obtained after a cell was lysed by alkaline lysis compared with cell lysis by proteinase K/sodium dodecyl sulphate or freezethawing in liquid nitrogen (unpublished data), the alkaline lysis method was subsequently used on single blastomeres and before ß-globin gene amplification. A 5 µl aliquot of lysis buffer (200 mmol/l KOH, 50 mmol/l dithiothreitol, pH 9.5) was added to each sample. The samples were heated for 10 min at 65°C. Subsequently, the alkaline lysis buffer was neutralized by the addition of a neutralizing buffer (900 mmol/l TrisHCl, pH 8.3, 300 mmol/l KCl, 200 mmol/l HCl) (Cui et al., 1989).
After cell lysis, the ß-globin gene of all samples containing biopsied blastomeres was amplified by nested PCR.
Nested PCR
The first and second PCR mix contained PCR buffer (2.0 mmol/l MgCl2, 10 mmol/l TrisHCl, pH 8.6, 50 mmol/l KCl, 0.01 w/v gelatin), 200 µmol dNTP (Boehringer Mannheim, Germany), 100 pmol primers (HPLC purification grade, Pharmacia, Sweden) and 0.5 U Taq polymerase (Perkin Elmer, USA). PCR mix 1 contained the outer primers, and PCR mix 2 contained the inner primers (Table II
). The outer and inner primers were used in everyday routine of PND application (Maggio et al., 1993
). PCR mix 1 was added to each Eppendorf tube containing a single blastomere in order to obtain a 100 µl total reaction volume. Each sample was put into a DNA thermal cycler (GeneAmp PCR System 2400, Perkin Elmer, USA). The programme of the PCR reaction 1 was 5 min at the initial 94°C denaturation temperature, followed by 30 cycles of 60 s at 94°C (denaturation), 60 s at 55°C (annealing), 60 s at 72°C (extension) and a final extension step at 72°C for 10 min. A 5 µl aliquot of the first PCR was added to 95 µl of PCR mix 2 and was run on the PCR programme 2: 5 min at 94°C followed by 40 cycles of 60 s of denaturation at 94°C, 60 s of annealing at 55°C, 60 s of extension at 72°C and a final extension step at 72°C for 10 min. A portion (15%) of the second PCR product was run on a 2% agarose gel in 0.5X Tris-borate/EDTA buffer stained with 0.5 µg/ml ethidium bromide.
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Genetic diagnosis
The ß-globin gene mutations diagnosed were: Cd39 (CT), IVS1 nt1 (G
A), IVS1 nt6 (T
C) and IVS1 nt110 (G
A) and HbS (sickle cell mutation). The genetic diagnosis of amplified samples was performed by enzymatic digestions for the corresponding mutations and reverse dotblot, except for IVS1 nt110 mutation where only reverse dotblot analysis was available.
Enzymatic digestions
HbS and IVS1 nt6 mutations
Samples (17.5 µl) of the PCR reaction product were digested by the addition of 5 U restriction enzyme DdeI (Boehringer Mannheim) for the HbS mutation or SfaNI (Biolabs, USA) for the IVS1 nt6 mutation and 2 µl of buffer 10X. The reaction tube was incubated for 3 h at 37°C.
IVS1 nt1 mutation
Samples (17.5 µl) of the PCR reaction product were digested by the addition of 5 U restriction enzyme BsaBI (Biolabs) and 2 µl of buffer 10X. The reaction tube was incubated for 3 h at 60°C.
Cd39 mutation
Samples (14 µl) of the PCR reaction product were digested by the addition of 3 U restriction enzyme MaeI (Boehringer Mannheim) and 16.5 µl of buffer 2X. The reaction tube was incubated for 3 h at 45°C.
It had been verified previously that the enzymatic digestions were complete when the incubation period was 3 h. The enzymatic digestion product was run on 3% ultrapure agarose gel (Gibco BRL, UK) and stained with 0.5 µg/ml ethidium bromide.
The wild-type allele is noted HbA. Figure 2 shows the corresponding restriction enzyme patterns on normal and mutated alleles for each mutations.
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To perform the reverse dotblot, a second PCR reaction mix was prepared which contained 200 µmoles dNTP and biotin-16-dUTP. The hybridization to amino-modified oligonucleotides for ß-thalassaemia and sickle cell anaemia and colour detection have been described elsewhere (Maggio et al., 1993).
Non-transferred and surplus embryos
The embryos that were biopsied and diagnosed but not transferred (defined `non-transferred embryos') and the surplus embryos that were not available for biopsy (slow embryo development or highly fragmented; defined as `surplus embryos') were donated to research programmes with the written consent of the couple. The non-transferred embryos were transferred into a 0.2 ml Eppendorf tube containing 2 µl of sterile PBS solution and their genetic diagnosis was compared with the previous blastomere analysis. When possible, one or two blastomeres were biopsied from the surplus embryos. As previously described, the blastomere and remaining embryo genotypes were determined and compared among them.
Definitions
Amplification efficiency is the number of samples with amplification of one or both alleles on all amplification samples. Amplification failure is the non-amplification of both alleles of a single cell. ADO is the absence of amplification of one of the two alleles of a single cell and is calculated on the number of amplified samples.
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Results |
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One blastomere and the whole embryo of the surplus embryos were lysed and diagnosed. From one surplus embryo of couple C', one biopsied blastomere was diagnosed as IVS1 nt1/IVS1 nt1. This was an aberrant diagnosis, and the whole-embryo diagnosis resulted in HbA/IVS1 nt1. From one surplus embryo of couple E, one biopsied blastomere was diagnosed as HbA/HbA, while the whole-embryo analysis resulted in HbA/Cd39. The analyses of single biopsied blastomeres were confirmed by whole-embryos diagnoses in 14 surplus embryos with slow or irregular in-vitro development.
The ß-globin gene analysis of non-transferred (denoted NT) embryos and surplus (denoted S) embryos confirmed by blastomere analysis are summarized in Table V.
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In total, 31 embryos during PGD and 16 surplus embryos were successfully analysed. Among all 47 embryos analysed, 18 (38%) were homozygous for wild-type allele, 18 (38%) were heterozygous for one wild-type allele and one mutated allele, and 11 (23%) had two mutated alleles. No amplification of the negative controls was observed.
Clinical data after PGD and prenatal analysis
At 12 days after embryo transfer, five positive ß-HCG tests were reported. Four clinical pregnancies were established, and after 7 weeks, five gestational sacs with five heart beat fetuses (2+1+1+1) were observed by ultrasound scanning. One pregnancy with one fetus miscarried at 11 weeks, while all other pregnancies underwent CVS after 11 or 12 weeks for confirmation of PGD for ß-thalassaemia (and sickle cell disease for couple G). The CVS results and miscarriage analysis confirmed the preimplantation genetic analysis (see Table IV). The twins were born at 34 weeks, and the two single babies were born at 38 weeks by Caesarean section. All the babies were healthy at birth.
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Discussion |
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On a world-wide scale, PGD for ß-thalassaemia and/or sickle cell anaemia has already been applied on single blastomeres (Kanavakis et al., 1999; Xu et al., 1999
; De Rycke et al., 2001
), and on the first and second polar bodies (Kuliev et al., 1998
, 1999
). The molecular strategies used were DNA amplification followed by genetic diagnosis by denaturing gradient gel electrophoresis analysis (Kanavakis et al., 1999
), restriction enzyme digestion (Kuliev et al., 1998
, 1999
; De Rycke et al., 2001
), the creation of a new restriction enzyme recognition sequence involving the IVS1 nt110 mutation (Kuliev et al., 1998
, 1999
; De Rycke et al., 2001
) and the use of fluorescence PCR (De Rycke et al., 2001
). The amplification efficacy reached 73% on blastomeres from good-grade embryos (Kanavakis et al., 1999
), and 93% after fluorescence PCR according to others in their clinical application (De Rycke et al., 2001
). For the same authors, the ADO rate reached 19 and 012% respectively.
In the present study, PGD was applied clinically for ß-thalassaemia and sickle cell anaemia on two fertile carrier couples with previous experiences of therapeutic abortion for affected fetuses, and on five infertile carrier couples. In total, nine PGD cycles were carried out. After ovarian stimulation, ICSI and embryo culture to day 3, embryo biopsy was successfully performed on 32 embryos out of the 33 available, and 25 of these developed in vitro on day 4. Thirty-five of the 36 samples containing one or two blastomeres were successfully amplified by nested PCR. By adding this data to surplus embryos that were not selected for embryo transfer, an amplification efficacy of 98.1% was obtained. In addition, five pregnancies were obtained, of which one was biochemical and four were clinical. The CVS in the first trimester confirmed a twin pregnancy with one homozygous for wild-type fetus, and one heterozygous fetus for Cd39 ß-globin mutation, and two singleton pregnancies (one wild-type fetus and one heterozygous fetus for IVS1 nt1 ß-globin mutation). The fourth pregnancy stopped on the 11th amenorrhoeic week, and the genetic analysis of abortion material revealed a homozygous fetus for wild-type allele as diagnosed at the preimplantation stage. Adding these results to those of the selected embryos that were non-transferred and the surplus embryos with slow or irregular development in vitro, it could be concluded that ADO occurred three times among embryos; this indicates an ADO rate of 8.6%. It is important to note that reverse dotblot and restriction enzyme digestion results were mutually confirmatory in the genetic analysis for all samples containing one or two blastomeres, and that reverse dotblot was used here for the first time in PGD for ß-thalassaemia and sickle cell anaemia.
The first results obtained with the clinical application of PGD for ß-thalassaemia and sickle cell anaemia were encouraging and resulted in four clinical pregnancies. Today, an increasing number of carrier couples attend the authors' centre from different regions of Italy. PGD is a good alternative for fertile couples who recognize in this technique a serious chance of avoiding pregnancy termination of an affected fetus following positive PND. It is also becoming evident that PGD appeals to young at-risk couples that have never attempted to achieve a pregnancy for fear of therapeutic abortion. These couples prefer to undergo IVF in order to eliminate the risk of transmitting ß-globin disorders.
The molecular protocol of amplification presented here leads to the diagnosis of 86.2% of ß-globin gene mutations found throughout Sicily (HbS, Fr cd6, Fr cd8, IVS1 nt1, IVS1 nt2, IVS1 nt5, IVS1 nt6, IVS1 nt110, IVS1 nt116, IVS1 nt130, Cd30, Cd39, Fr cd44) (Giambona, 1995). In the present study, PGD was applied for HbS, IVS1 nt1, IVS1 nt6, IVS1 nt110 and Cd39 mutations that represent 6.9, 8.3, 12.6, 23.4 and 33.9% respectively of ß-globin gene mutations present in the Sicilian population. Currently, molecular protocols are being set up for other ß-thalassaemic mutations further away from the amplified region presented here (e.g. 87, IVSII nt745). Nowadays, fluorescence PCR is becoming the method of choice in the clinical application of PGD for single gene disorders as it provides greater reliability and accuracy, less ADO and a time reduction for genetic analysis compared with traditional techniques such as nested PCR and enzymatic digestion by restriction enzymes (Findlay et al., 1995
, 1999
; Ray et al., 1996
; Sermon et al., 1998
; Sherlock et al., 1998
). Furthermore, modifications such as multiplex fluorescence PCR (Kuliev et al., 1998
; Rechitsky et al., 1998
; Findlay et al., 1999
; Ioulianos et al., 2000
; Eftedal et al., 2001
; Piyamongkol et al., 2001
) make the diagnosis of different DNA regions possible on the same single cell and keep ADO at a minimum level. It has been demonstrated (Lewis et al., 2001
) that for recessive disease, the highest probability of transferring an available embryo with the minimum risk of misdiagnosis is obtained by genotyping separately two single blastomeres for the single gene disorder and a linked marker (polymorphism, microsatellite markers). It is most likely that these guidelines will be followed for further clinical applications.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 31, 2001; accepted on November 30, 2001.