Molecular Human Reproduction, Vol. 9, No. 10, 631-638,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases
Submitted on April 1, 2003; resubmitted on June 11, 2003. accepted on June 18, 2003
1 Department of Obstetrics and Gynecology, Royal Victoria Hospital, Montreal, H3A 1A1, Quebec, 2 Department of Experimental Medicine, McGill University, Montreal, H3A 1A1, Quebec, 3 Montreal Neurological Institute, Montreal, H3A 2B4, Quebec, 4 Department of Human Genetics, McGill University, Montreal, Quebec, Canada and 5 The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA 23507-1627, USA
6 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, McGill University, Montreal, H3A 1A1, Quebec, Canada. e-mail: asangla.ao{at}muhc.mcgill.ca
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Abstract |
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To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6–8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.
Key words: heteroplasmy/mitochondrial DNA/mtDNA diseases/mouse model/preimplantation genetic diagnosis
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Introduction |
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Inherited mitochondrial DNA (mtDNA) diseases are conservatively estimated to affect
1 in 8500 individuals (Chinnery and Turnbull, 2001). Dysfunction in the mitochondrial respiratory chain causes a diverse group of incurable, progressive diseases, both in adults and children, which often lead to severe disability and premature death. This heterogeneous group of diseases can involve a wide variety of organs and systems, especially the central nervous system, skeletal and cardiac muscles, pancreas, liver and kidney (DiMauro and Schon, 2001).
Mitochondrial diseases can result from mutations in mitochondrial genes encoded either in the nuclear DNA or the mitochondrial genome. The mammalian mitochondrial genome is a small (16.5kb) double-stranded circular DNA that is maternally inherited. Each mitochondrion contains multiple copies of mtDNA, and every somatic cell contains several hundred to thousands of copies (Nass, 1969; Bogenhagen and Clayton, 1974; Satoh and Kuroiwa, 1991). The mitochondrial genome codes for 37 genes, including genes for 22 tRNA, two rRNA and 13 polypeptides of the mitochondrial respiratory chain. More than 100 mtDNA mutations have been characterized both in protein-coding and tRNA and rRNA genes (DiMauro and Schon, 2001).
The processes of mtDNA transcription and replication differ from that of nuclear DNA. Of particular relevance to mtDNA disease is that mtDNA replication is not rigidly coupled to the cell cycle (Clayton, 1982). During mitosis, mtDNA replicates so that daughter cells receive similar copy numbers, but some templates may replicate more than once during any cell cycle, others not at all. mtDNA is randomly partitioned to the daughter cells at cytokinesis. Germ line mutations in mtDNA can lead to the co-occurrence of two or more sequence variants in a cell (mtDNA heteroplasmy). These sequence variants will randomly segregate when transmitted through the maternal germline. A genetic bottleneck for mtDNA occurs in the primordial germ cells, which results in rapid segregation of mtDNA sequence variants and thus a wide variation in the relative proportions of the two or more types of mtDNA in the oocytes of a heteroplasmic mother (Jenuth et al., 1996; Chinnery et al., 2000).
The different pattern of transmission seen in diseases affecting maternally inherited mtDNA, as compared with disorders caused by mutations of nuclear DNA, can be explained by this rapid stochastic segregation of pathogenic mtDNA mutations between generations. Inherited pathogenic nuclear DNA mutations are almost always present in the same number in every cell of the body (one copy for dominant and X-linked conditions and two copies for recessive disorders), and their presence will lead to the manifestation of a phenotype. However, pathogenic mtDNA mutations can be present in any cell or tissue at levels varying from 0 to 100%, and do not express a phenotype until the mutant levels exceed 85%, the threshold beyond which cellular dysfunction will become evident (Boulet et al., 1992). This threshold can vary according to the particular mutation and the energy needs and characteristics of individual organs and tissues (Hayashi et al., 1991; Dubeau et al., 2000; Thorburn and Dahl, 2001). Most individuals carrying a pathogenic mtDNA mutation are heteroplasmic for mutant and wild-type mtDNA, and those with mutant levels below the threshold for disease expression will not present with a clinical phenotype. Nevertheless, a heteroplasmic woman carrying a mtDNA mutation, who does not express a phenotype, can transmit considerably higher levels of mutant mtDNA to her offspring, at or above the threshold that could lead to the manifestation of a clinical disease.
One way such a woman can reduce the risk of giving birth to an affected child is to measure the mutant load in the chorionic villi or amniocytes by performing prenatal testing. Initially there was concern as to whether the level of mutant mtDNA detected in fetal cells at 10–18 weeks gestation could predict the mutant load in other tissues at birth. However, current data from animal and human studies suggest that there is little tissue variation or selection operating on mtDNA mutations in utero (Jenuth et al., 1997; White et al., 1999; Chinnery et al., 2000; Dahl et al., 2000). Therefore, the levels found in prenatal samples can be used to predict the mutant load in most tissues at birth. Prenatal diagnosis has been reported occasionally for mtDNA mutations affecting protein-coding genes but is not widely used (Harding et al., 1992; Thorburn and Dahl, 2001).
Prenatal diagnosis is only available once a pregnancy is established. If the couple wish to prevent the birth of an affected child they will need to decide whether or not to terminate a fetus with a high proportion of mutant mtDNA, or even one with an intermediate mutant load. Recently a new approach to antenatal testing, preimplantation genetic diagnosis (PGD), has been introduced that takes advantage of techniques developed to analyse DNA from single cells. PGD, used in conjunction with IVF, can diagnose specific genetic disorders by PCR, using the polar body from an oocyte or one or two cells from a preimplantation embryo (Handyside et al., 1990; Verlinsky et al., 1990; Ao and Handyside, 1995). This approach has the advantage of providing a diagnosis before a pregnancy is established and eliminates the dilemma of whether or not to terminate a pregnancy. The analysis of the first polar body from an unfertilized oocyte may be preferred by some couples with strong reservations about embryo testing. Unlike Mendelian disorders, where the paternal component can also cause disease, only the maternal mitochondrial contribution needs to be determined for mtDNA diseases. Therefore, polar body analysis is theoretically suitable for diagnosing this group of diseases (Briggs et al., 2000). The second approach of blastomere biopsy, which is more widely used for single gene defects for PGD, allows for the detection of both parental genomes. Blastomere biopsy also avoids the testing of material that may not have the potential to fertilize.
Before PGD can be considered for mtDNA diseases, it is important to ascertain that the proportional levels of mutant to wild-type mtDNA quantified in the biopsied cell are representative of the levels in the embryo as a whole. Once this is assured, PGD could be carried out for inherited human mtDNA diseases and only embryos with undetectable or very low amounts of mutant mtDNA would be regarded as suitable for transfer. It is, however, currently unknown whether mutant and wild-type mtDNA are distributed equally between the ooplasm of an oocyte and its polar body, or between the blastomeres of an early cleavage stage embryo.
Difficulty in obtaining oocytes or embryos from women with mtDNA mutations makes the use of appropriate animal models very important in investigating the segregation of mtDNA during early cleavage development. For our experiments we used a mouse model, which is heteroplasmic for two polymorphic mtDNA sequence variants (NZB and BALB) (Jenuth et al., 1996). The transmission of the two mtDNA genotypes to offspring in these mice has been shown to be a stochastic process determined by random genetic drift. In the present study the heteroplasmic animals were used to investigate the distribution of the two genotypes of mtDNA in female gametes and early cleavage stage embryos. The segregation of the two mtDNA sequence variants was measured between the ooplasm and first polar body of unfertilized mature oocytes, and between blastomeres of early cleavage stage embryos at different stages of development. The results of these experiments are discussed in the context of the feasibility of clinical PGD as a method to prevent inherited mtDNA disorders.
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Materials and methods |
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mtDNA genotype analysis of female animals
Heteroplasmic animals were generated as previously described (Jenuth et al., 1996). These mice are heteroplasmic for NZB and BALB mtDNA, two genotypes that differ in 101 nucleotides, predicting 15 amino acid substitutions. mtDNA was genotyped using a restriction fragment length polymorphism method. An RsaI site at position 3691 in the ND1 gene present in BALB but absent in NZB mtDNA was used to genotype individual animals. The mtDNA genotype was determined on a PCR-amplified fragment encompassing the polymorphic site. Tail biopsies were taken from anaesthetized female mice and the genomic DNA was extracted by standard procedures (Sambrook et al., 1989). Total genomic DNA was amplified using the following primers: forward MT9 nt 3571–3591, 5'-GAGCATCTTATCCACGCTTCC-3'; reverse MT10 nt 4079–4059, 5'-CTGCTTCAGTTGATCGTGGGT-3'. The PCR and cycling conditions were as previously described (Jenuth et al., 1996). Briefly, PCR reactions were carried out in a total volume of 50 µl with 1xpotassium-free PCR buffer (100 mmol/l Tris–HCl, pH 8.3), 2.5 mmol/l MgCl2, 200 µmol/l each dNTP, 0.4 µmol/l each of MT9 and MT10 primers and 1.25 IU of Taq polymerase (Gibco, Canada). The PCR was carried out on a Perkin Elmer 9600 thermocycler using the following programme: 5 min denaturation at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C. In the last cycle 1.5 µCi of [
-32P]dCTP was added to the reaction to radiolabel the PCR product. A 15 µl aliquot of this reaction was digested with 10 U of RsaI overnight at 37°C and run on a 10% non-denaturing polyacrylamide gel.
Isolation of oocytes and embryos
Ovulated metaphase II oocytes were obtained from mice between 6 weeks and 4 months of age. This work was approved by the McGill University Animal Care Committee. Females with known levels of heteroplasmy were superovulated by i.p. injections of 7.5 IU of pregnant mares’ serum gonadotrophin (Sigma, Canada) followed by 5 IU of hCG (Sigma) 44–48 h later. Sixteen to 18 h later the oviducts were removed and the oocytes released by tearing the swollen ampulla region of the oviduct into HEPES-buffered potassium simplex optimized medium (H-KSOM). The cumulus cells surrounding the oocytes were removed by brief incubation in hyaluronidase (300 µg/ml). To obtain early cleavage stage embryos, superovulated mice were mated with stud males overnight and checked for the presence of a plug the following morning. Two-, 4- and 8-cell embryos were obtained by flushing the oviducts of pregnant females on days –1.5, –2 and –2.5, respectively. When more than five 2-cell embryos were available, some were disaggregated at that stage and the remainder were cultured in vitro for 1 or 2 days to allow further cell division before they were disaggregated. All embryos were cultured in pre-equilibrated fresh KSOM medium under paraffin oil at 37°C in 5% CO2 in air.
Cell collection
The zona pellucida was removed from oocytes and embryos by a short incubation in acid Tyrode’s solution (pH 2.5) and washed in H-KSOM. The first polar body from each oocyte was carefully aspirated into a fine pulled glass pipette and transferred to a PCR tube containing 5 µl of alkaline lysis buffer (Cui et al., 1989). The ooplasm was then pipetted into a separate lysis buffer tube using a clean pulled glass pipette. For the embryos, each zona-free embryo was incubated for 5 min in Ca2+- and Mg2+-free media to allow decompaction of blastomeres, and then the cells were disaggregated by gentle aspiration and expulsion from a pulled glass pipette. Each blastomere was then pipetted into a separately labelled lysis buffer tube. Each cell was lysed by heating the PCR tube to 65°C for 15 min and 5 µl of neutralizing buffer were added (Cui et al., 1989).
mtDNA genotype PCR of oocytes, polar bodies and blastomeres
The amount of lysed cell sample necessary in each reaction mix was calculated from pilot data and depended on the size of the initial cell. For oocytes 1 µl was used, for polar bodies 3 µl and for cleaved blastomeres 2 µl were used. In each experiment a negative control consisting of 5 µl of double-distilled H2O was included. Duplicate samples of each lysed cell underwent a PCR assay to amplify the fragment encompassing the polymorphic site as described for genotyping the female animals except that 35 rather than 30 cycles were used. Overall, 131 PCR reactions were necessary to analyse the 58 oocyte and polar body samples and 448 PCR reactions were necessary to analyse the 211 blastomeres.
Analysis of results
Gels were analysed on a Storm PhosphorImager (Molecular Dynamics). The percentage NZB mtDNA was calculated for each reaction mix by adding the amount of radioactivity in bands 2 and 4 and dividing this by the value in band 1 (see Figure 1). The product in band 3 is common to both types of mtDNA. The mean %NZB mtDNA was deduced, for each cell, from the relative amount of NZB mtDNA in each duplicate and the coefficient of variation (CV) was calculated (see Figure 1 and Table I). The resulting mean %NZB for each cell was considered to be a reliable value when the CV of duplicate samples was <10. When the CV of the duplicates was >10, a triplicate sample of the cell underwent PCR, and if this reduced the CV to <10 the results from that cell were considered valid. However, if the CV still remained high, the experimental error was considered too great and the results from the whole oocyte or embryo were considered as unreliable and were excluded from the study.
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Results |
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To compare the distribution of heteroplasmy between the ooplasm of an oocyte and its corresponding polar body, 29 mature oocytes were successfully collected from six mice with maternal levels of heteroplasmy ranging from 17.0 to 67.7%. In addition, eight oocytes were not suitable for the study, of which five had a germinal vesicle present and the other three were either at the metaphase I stage or the polar bodies had completely disappeared. Overall, 58 oocyte and polar body samples were obtained for analysis. The results were analysed as illustrated in Table I, which shows the raw data from two representative oocytes and their corresponding polar bodies obtained from female mice with different levels of heteroplasmy. This yielded closely duplicated results in 22 oocytes with their corresponding polar bodies and the exclusion of seven oocytes where the results from one or both cells tested did not produce results, or the results did not satisfy the criteria stated in Materials and methods. The average number of oocytes, which gave full results, from each mouse, is 3.7 (range 1–7). The cumulative data from all oocyte and polar body pairs are presented in Table II and Figure 2. The coefficient of determination (r2) for the levels of heteroplasmy in the ooplasm of the oocytes compared with those in the polar bodies was 0.99 (Figure 2a), whilst the value for the level of heteroplasmy in the gamete (the mean value in the ooplasm and polar body) compared with the maternal genotype was 0.32 (Figure 2b). Within each gamete, the difference in the levels of heteroplasmy in the ooplasm compared with its respective polar body ranged from 0.1 up to 6.1% (Table II).
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When the levels of heteroplasmy in the blastomeres of cleaved embryos at different developmental stages were investigated, individual cells from 55 embryos were successfully collected from 15 female mice. Levels of heteroplasmy in the mice ranged from 7 to 74% and the levels in the embryos ranged from 3 to 73%.
A cleaved embryo was considered for inclusion in the study only when >50% of the blastomeres could be individually collected. The results from two embryos were excluded as one or more cells tested did not give closely duplicated results. Therefore, closely duplicated results for all cells tested were obtained in 53 embryos at different cleavage stages (see Table III). There were 19 2-cell embryos, 18 4-cell embryos and 16 6–8-cell embryos. The average number of embryos giving a full result from each mouse was 3.5 with a range of 1–13.
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If a small cohort of 2-cell embryos was obtained from one mouse, the embryos were disaggregated and cells collected on the day of retrieval. If a larger cohort of 2-cell embryos was obtained, the cells from a proportion of the embryos were collected on the first day and the remainder left in in-vitro culture until the following day to allow further division. This acted as an experimental control to ensure that the distribution of heteroplasmy mtDNA was independent of embryonic cleavage stage.
The data presented in Table III show the range of levels of heteroplasmy detected within all the cells of a particular embryo. This ranges from 0% difference between the cells within each embryo to the largest difference of 6%. The median difference in all 53 embryos was 2%. Within the cohort of embryos obtained from each mother, a wide variation in the levels of heteroplasmy could be observed between embryos (see Table III). However, the mean level of heteroplasmy seen in a group of embryos from one mouse was similar to that seen in the mother (median difference = 3.6, r2 = 0.94). This relationship was even stronger (median difference = 2.75, r2 = 0.96) when only those mice (of which there were eight) with three or more embryos were included.
From the mature oocytes, 24.0% (7/29) failed to give a result in either one or both of the cells. This comprised five polar bodies, one ooplasm and one total oocyte (both ooplasm and polar body). The failure rate for the polar bodies alone was 20.7% (6/29). For the cleaved embryos, two embryos failed to give a result. One was a 2-cell embryo in which one cell failed to give a result; the other was a 6-cell embryo in which two cells did not provide any result. Therefore, the failure rate in the case of blastomeres was 1.4% (3/211). For the oocytes included in the study, it was also necessary to run triplicate samples to verify the results for 13/22 (59.1%) polar bodies and for 2/22 ooplasm samples. Of the 203 blastomere samples included in the study, five triplicate samples had to be run for 2-cell embryos, two for 4-cell and 19 for 6–8-cell embryos, giving a total of 26/203 (12.8%).
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Discussion |
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Couples at high risk of an inherited genetic disease have the option of prenatal diagnosis and of a therapeutic termination of the pregnancy if the fetus is affected. An alternative method of diagnosis for a number of these diseases is through PGD, and an expanding number of single gene defects have been diagnosed using this technique (ESHRE PGD Consortium, 2002; Ao, 2003). Unlike preimplantation genetic analysis for Mendelian disorders, where the presence or absence of the specific mutation is detected from the nuclear DNA, PGD for mtDNA diseases aims to detect the proportion of mutant mtDNA in the embryo. Therefore, before PGD for mtDNA diseases can be offered it is important to ensure that the segregation distribution of mutant mtDNA through the female germline is understood. One approach available to study this transmission, especially in view of the rarity of suitable human clinical material, is the use of an appropriate animal model. Mouse strains have been constructed which are heteroplasmic for two neutral mtDNA polymorphisms (Jenuth et al., 1996; Meirelles and Smith, 1997). Jenuth and colleagues demonstrated that these mtDNA polymorphisms were transmitted to the offspring with levels of heteroplasmy largely determined by random genetic drift, similar to what has been observed in many human mtDNA diseases (Jenuth et al., 1996; Chinnery et al., 2000). We used these heteroplasmic mice to determine the segregation pattern of the two neutral genotypes of mtDNA in early embryos in order to evaluate the potential of PGD for human mtDNA disease.
PGD can be performed on a polar body from an oocyte or zygote, on a blastomere from a cleavage stage embryo, or on a few trophectoderm cells from a blastocyst. First polar body biopsy, from an oocyte, has certain advantages in that it circumvents the ethical issues concerned with the manipulation of human embryos and allows an extended period of time available for genetic analysis before the transfer of cleavage stage embryos. In our mice, the distribution of the two types of mtDNA was found to segregate evenly between the ooplasm and the first polar body of each mature unfertilized oocyte. The high value of the coefficient of determination verified that the proportion of mtDNA genotypes detected in polar body and ooplasm of a mature oocyte were almost identical. This demonstrates that the small number of mitochondria present in the polar body are representative of the entire oocyte. However, the mean level of heteroplasmy in the oocyte (from the ooplasm and polar body combined) was not similar to that of the maternal genotype, due to the genetic bottleneck for mtDNA transmission (Jenuth et al., 1996). Also of note is that mice with different maternal heteroplasmy had different distribution patterns in their oocytes. The maternal genotypes from the cohort of mice studied coincidentally fell into three groups of heteroplasmy (68%, 40–41% and 17% NZB mtDNA) (Table II). The 10 oocytes from the two mice with 68% NZB mtDNA showed a range of heteroplasmy distributed either side of the maternal genotype, similar to the distribution seen in many human mtDNA mutations (Chinnery et al., 2000). The two mice with 40 and 41% heteroplasmy gave six and three oocytes, respectively. Although both mothers had similar proportions of mtDNA, all the oocytes from one mouse had higher levels of heteroplasmy than the maternal genotype (from 56 to 78%) whereas all the oocytes from the other mother had lower levels (from 5 to 22%). The two mice with the lowest levels of heteroplasmy (17%) had low levels of heteroplasmy in all the oocytes tested. These findings are likely to be due to the small cohort of gametes collected from each mouse rather than any selection of one mtDNA genotype. Data from published human pedigrees support the notion that transmission of pathogenic mutations is random as in the mouse model, although a skewed mtDNA segregation has been suggested in one woman whose oocytes all had either extremely high or almost zero levels of mutant mtDNA (Blok et al., 1997).
The most commonly used technique for PGD is the biopsy and analysis of cleavage stage embryos (Ao et al., 1996; Grifo et al., 1998; Kanavakis et al., 1999; Vandervorst et al., 2000; ESHRE PGD Consortium, 2002; Harper et al., 2002; Pickering et al., 2003). Using our mouse model, it was found that the distribution pattern of heteroplasmy within the blastomeres of each cleaved embryo was identical through each cleavage stage examined, which is consistent with our preliminary results (Molnar and Shoubridge, 1999). Although the cytoplasmic volumes of blastomeres from embryos at the 2-, 4-, 6- and 8-cell stage are different, the ability to detect the proportion of the two mtDNA genotypes was not affected. There is reported to be an asymmetrical mitochondrial distribution at the pronuclear stage of human embryos that could lead to varying mitochondrial complements in different blastomeres (Van Blerkom et al., 2000). However, unless it is extreme, variance in the total number of mitochondria present in a blastomere should not affect the overall proportion of different genotypes of mtDNA observed in each cell. Similar to the results observed in oocytes, there was a wide variation in the proportions of mtDNA in cleaved embryos compared with the levels in the mothers. For example, a maternal genotype of 47% NZB mtDNA produced embryos with 18–73% heteroplasmy (see Table III).
We have explored the potential of polar body and blastomere analysis in performing preimplantation diagnosis for inherited mtDNA diseases in two groups of patients, (i) those who are opposed to any manipulation after fertilization and (ii) those amiable to embryo biopsy, to assess which method permitted greater reliability of diagnosis. Both approaches gave equal accuracy in detecting the percentage mtDNA genotypes present in the polar body of an oocyte compared with the ooplasm, and in one blastomere compared with the remaining cells of the embryo. Although the levels of heteroplasmy between oocytes ranged from 1 to 78%, as expected, the range of difference between each polar body and its respective ooplasm was from 0.1 to 6%. Accordingly, the difference in the detected levels of heteroplasmy between blastomeres of an early cleavage stage embryo gave a median of 2% and a range of 0–6%. This accuracy of diagnosis from the first polar body or blastomere would allow for the reliable selection of unaffected embryos and the avoidance of a misdiagnosis. However, cleavage stage biopsy may allow more accurate determination of the exact proportion of mutant mtDNA load in the resulting fetus if, as suggested from IVF failed-to-fertilize oocytes and poor quality embryos, human embryos have a significantly reduced level of mtDNA rearrangements than oocytes (Barritt et al., 1999).
Although both methods of biopsy permitted accurate results, the genetic analysis of blastomeres showed a higher efficiency of diagnosis. The rate of failure to obtain a result was almost 21% in polar bodies compared with <2% in blastomeres. The results from the polar bodies were also less consistent in that more triplicate samples needed to be run to verify the levels of heteroplasmy in a polar body compared with a blastomere (59 to 13%, respectively). The poorer amplification of polar bodies compared with blastomeres could be due to the difference in cytoplasmic volumes, and therefore the numbers of mitochondria, in the two cell types. A blastomere from an 8-cell embryo contains 12.5% and a polar body 0.5% of the total ooplasmic volume (Briggs et al., 2000). Therefore, a blastomere would be expected to have a higher copy number of mtDNA (12 500 copies), which could result in more efficient PCR amplification. In humans, a wide variation in the mtDNA copy number can exist between mature oocytes even from the same patient (Steuerwald et al., 2000; Reynier et al., 2001; Barritt et al., 2002). However, our PGD protocol is based on the proportions of each mtDNA genotype, and detection of a particular genotype is accurate with as few as 10 copies present. Therefore, a low copy number would not affect the sensitivity of our test or interpretation of the result. It is also possible that the mtDNA in the polar body has degenerated compared with the mtDNA in the blastomere of a developing embryo. It has been shown that the first polar body in both mouse and human oocytes undergoes a rapid degeneration process during in-vitro incubation (Munné et al., 1995; Choi et al., 1996). Efficient diagnosis will result in the availability of more unaffected embryos, allowing a better selection of morphologically good quality embryos for transfer and thereby increase the success rate in any given treatment cycle.
The interpretation of predictive data after PGD for mtDNA diseases could employ similar methods to those used in prenatal diagnosis. For most mtDNA mutations, a mutant load of 30% or >90% can be interpreted as having low or high probability, respectively, of an affected outcome. Intermediate mutant loads would represent a ‘grey area’ in which interpretation would be difficult. In genetic prenatal testing, an intermediate mutant load would indicate that the fetus could be phenotypically affected, and this would lead to a dilemma of whether or not to terminate the pregnancy. From the available data it is predicted that embryos carrying zero or low mutant loads, as diagnosed by PGD, would be regarded as suitable for embryo transfer and embryos diagnosed with intermediate loads would not be transferred. This may reduce the number of embryos available for transfer but would not be as serious as the possible termination of a pregnancy at risk of manifestation of a mtDNA disease but which may be unaffected. However, it is possible that the presence of a low proportion of mutant mtDNA in the embryo may not indicate the exact mutant load in the resulting individual as significant tissue-specific segregation can occur in some mtDNA mutations (Poulton and Morten, 1993; Weber et al., 1997).
Patients requesting PGD are generally fertile but are required to undergo an IVF cycle involving hormonal stimulation, operative procedures and financial costs, similar to an infertile patient. Therefore, PGD for mtDNA diseases may be more suitable for women carrying higher proportions of mutant mtDNA, who have a strong likelihood of having a fetus with an intermediate or high mutant mtDNA load. It is thus likely that these women will have a large proportion of oocytes with a substantial mutant load and they may require multiple cycles of ovarian stimulation to achieve a healthy child. Even in such cases, the results from one cycle of PGD would provide valuable information for guiding subsequent reproductive choices. Collecting oocytes from a superovulated cycle and analysing them as a sample of the oocyte population as a whole has been used diagnostically for women with mtDNA deletions (Poulton et al., 2002). Although it is not certain that the levels in these oocytes represent the whole ovarian population, the amount of mutant mtDNA in superovulated oocytes would give a good indication of the likely content in any child. To date, no PGD cycle has been completed for any woman carrying a mtDNA mutation; although one patient with an A3243G mutation was reported as having undergone treatment, no embryo transfer was performed (ESHRE PGD Consortium, 2002).
Before offering PGD for mtDNA diseases, we need to be confident that the transmission of these neutral polymorphisms in our mouse model is reflective of the transmission seen in human mtDNA disease. Attempts to study the inheritance of mtDNA heteroplasmy in humans have been hampered by a number of problems, including the fact that very few human pedigrees are large enough to permit reliable calculations of the variance between offspring. Two groups, who looked at either the offspring or primary oocytes from women carrying different mtDNA mutations, have reported that there is no strong positive or negative selection for pathogenic mtDNA occurring in the oocyte or in early embryogenesis (Chinnery et al., 2000; Brown et al., 2001). They concluded that the predominant mechanism behind the transmission of pathogenic heteroplasmic mtDNA mutations is random genetic drift, as in our mouse model. However, it is possible that some point mutations or mtDNA rearrangements could exhibit different segregation behaviour (Chinnery et al., 2000).
In summary, we have shown that heteroplasmic mtDNA is distributed equally between a polar body and ooplasm of a mature oocyte, and also between the blastomeres of an early cleavage stage embryo in our mouse model. Transmission of heteroplasmic mtDNA in this murine system is similar to that seen in most reported human pedigrees segregating mtDNA mutations. Therefore, we conclude that PGD for inherited human mtDNA disorders is feasible; however, it should be approached with caution, as some pathogenic mutations could behave in a different manner. The biopsy and genetic analysis of one or two blastomeres from a cleaved embryo will give superior results as compared with the first polar body from an unfertilized oocyte in the diagnosis of mtDNA diseases. Finally, simple adaptation of the PCR protocol to avoid the need for radioactive labelling, with the use of real-time PCR or incorporating fluorescently labelled primers, would allow this diagnostic technique to be more widely applicable and may also further increase the efficiency of diagnosis.
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Acknowledgements |
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This paper received the Best Basic Science Paper Prize at the 48th Annual Meeting of Canadian Fertility and Andrology Society, Charlevoix, Quebec, Canada, September 25–28, 2002. The authors would like to thank Balthazar Lauzon for technical assistance. This research was supported by grants from the CIHR to E.A.S. N.L.D. is partially supported in the PhD programme by an academic award from the Department of Experimental Medicine. B.J.B. is supported by an NSERC postdoctoral fellowship. A.A. is generously supported by the McGill Reproductive Centre, Department of Obstetrics and Gynecology, McGill University. E.A.S. is an international scholar of the HHMI and a senior investigator of the CIHR.
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