Fig. 17.1
PGD cycles of the respective carrier females, performed in our center. Each cluster of bars represents an embryo with its tested blastomeres. The red dotted line represents the threshold level for transfer. For the Leigh carrier, the embryos in which the mutation was not detected are depicted as X. For these embryos, the numbers of analyzed blastomeres are not visible in the figure. ET embryo transfer, FR frozen, Bl blood, U urine, M muscle, H hair
Few additional reports of PGD performed for mtDNA disorders in other centers exist; a total of 12 cycles have been performed in nine mtDNA mutation carriers which resulted in the birth of five children [10, 31, 52, 54, 55]. In general, the mutation loads we observed for m.8993T>G among single blastomeres were concordant with previous reports [31, 52, 57]. Of note, interblastomere differences of 11 % have been noted [57] and fully descriptive data were not provided [52]; in our series, blastomeres/embryos with no mutation were overrepresented (25/28 embryos), making it difficult to draw a general conclusion. Interblastomere variation for the m.3243A>G mutation was generally larger and occurred more often than previously reported for this mutation [10, 54], although Monnot et al. did not perform single blastomere analysis for all embryos [10].
Vanderwoestyne et al. also reported large interblastomere variation of 24 % in an m.3243A>G embryo [53]. As interblastomere variation seems to occur more frequently in certain individuals, this itself might be a phenomenon subject to genetic factors [27] although insufficient data exist for such individual risk stratifications. All data taken together, nicely plotted in a figure by Steffann et al. [55], a generally homogeneous distribution of wild-type and mutant mtDNAs can be seen in individual human blastomeres regardless of the mutation, differing remarkably from data on artificially generated heteroplasmic macaque embryos [55, 58]. Based on human data which shows that single blastomeres can diverge, it is advisable to analyze two blastomeres instead of just one. The adverse risk of removing two cells from the embryo at biopsy is a negative influence on live birth delivery [59], illustrating the difficult balance between a safe and correct diagnosis on the one hand and optimizing the chance of pregnancy on the other.
Trophectoderm biopsy performed at the blastocyst stage provides a larger number of cells for analysis and appears to obviate the negative impact on live birth delivery. This approach would also enable more precise selection of a single embryo based on both mutation load and genetic sex. Male offspring with an mtDNA mutation will not encounter the risk of transmitting the mutation to their offspring. So far, only one blastocyst PGD for an mtDNA mutation (m.3243A>G) has been performed in humans although results were promising with regard to the applicability of blastocyst trophectoderm biopsy and PGD for mtDNA mutation carriers [54] (which had been supported by murine data [60]).
However, recently added follow-up data of the boy born after blastocyst PGD reported clinical symptoms and m.3243A>G mutant loads of 47 % and 46 % in blood and 52 % and 42 % in urine, respectively, at ages 6 weeks and 18 months [61]. The blastocyst mutation load had been only 12 % [54]. This contradicts the original report where no abnormal phenotype was reported and follow-up mutation load was 15 % in buccal mucosa at age 1 month; at ages of 5 and 12 months, the mutation load was measured by a commercial lab and found to be <10 % in blood and undetectable in buccal mucosa and urine [54]. While technical differences do exist between methods used to determine the mutation load, this cannot explain such a large difference. It is unclear what has happened, and the authors of both papers should work collaboratively to clarify this.
Data on the five children born so far after blastomere PGD at the 8-cell stage are much more reassuring [10, 27, 31, 52, 55]. Besides the balance between safety of embryo biopsy (the number of cells to remove for analysis) and subsequent reproductive outcome, the number of embryos available for analysis also brings some conflicting considerations. From the perspective of a cytogenetics laboratory, the more embryos available for study the better, since a larger sample improves the chances of having at least one embryo suitable for transfer (and thus improves the chances of delivering a healthy baby). However, there is a limit to the hormonal (over)stimulation that can be applied during IVF. and some clinically affected carriers will be found a priori to be poor candidates for PGD/IVF treatment (based on inacceptable health risks). For mtDNA mutation carriers approved to undergo IVF, it is important to realize that PGD for these indications represents a substantial risk reduction but not an absolute risk exclusion. This should be carefully discussed during patient counseling and the informed consent process. A 0 % mutation load only occurs seldomly (except for skewing mtDNA mutations). Furthermore, current data are suggestive, but not definitive, to guarantee that mutation load in the embryo stage will remain constant throughout life without passing the threshold level for symptoms at some later point. Nevertheless, we feel that for heteroplasmic mtDNA mutation carriers who want to have unaffected offspring who are biologically their own (and therefore not use donor oocytes), PGD represents the best therapeutic option at present. However, it should be acknowledged that PGD is not permitted in all jurisdictions.
PND Versus PGD: Specific Considerations with Respect to Skewing (8993) mtDNA Point Mutations
Although our considerations might be applicable to skewing mutations in general, only for the 8993 mutations do sufficient data currently exist. The characteristics of the nt8993 mutations make PND a feasible option for female carriers, particularly when mutation load is low. PGD is still an alternative in this group of mutation carriers with medium to high mutant load. The chance of producing embryos without the mutation is generally higher than for non-skewing mutations. In cases of high maternal mutation load, the majority of embryos is expected to have high mutation load although PGD will enable selection of those embryos with no or low mutation load. In contrast, PND would lead to the detection of multiple severely affected fetuses and recurrent pregnancy terminations.
If the maternal mtDNA mutation load is low, the majority of embryos would be expected to be without the mutation [27]. Due to the linear relationship between the mother’s and her offspring’s mutation load [29], for carriers with intermediate mutation load, the situation will be somewhere in the middle. In the choice between the two reproductive options and pregnancy risks, the burden of PGD treatment will need to be carefully considered.
Oocyte Donation
Perhaps the safest and most reliable method to prevent transmission of mtDNA disease is the use of donor oocytes accompanied by IVF using the partner’s sperm. However, the supply of suitable donors may be limited in some locations, and oocyte donation is not lawfully allowed in every country. Maternal relatives such as sisters will generally not be suitable as oocyte donors, as they are at risk of carrying the mutation in their oocytes as well. The latter cannot be excluded based on the absence of the mutation in blood or other tissues. An important personal reason for couples to reject oocyte donation is the fact that the resulting child would not be genetically related to the mother.
Nuclear Transfer
Nuclear transfer (maternal spindle transfer and pronuclear transfer) entails the transfer of the nuclear genome from an oocyte or zygote with mutated mtDNA in the cytoplasm (donor) to an enucleated acceptor oocyte or zygote of a healthy donor (acceptor) with presumably normal, mutation-free mtDNA. This technique is currently under investigation only in a research setting [62–67]. Although promising, the safety and efficacy of nuclear transfer which has been noted in primate models has yet to be shown compatible with humans, so this approach requires further study; important ethical issues also require resolution. Whether this technique will be able to completely exclude the risk of transmitting an mtDNA mutation or attain merely a reduction of this risk to offspring is still unclear, since nuclear transfer cannot avoid the co-transfer of small amounts (<1 % in spindle transfer) of mtDNA from the affected to the donor oocyte/zygote. Nuclear transfer techniques would offer a reproductive option for homoplasmic mtDNA mutation carriers and for heteroplasmic carriers with high mutation load, who might produce no or very few oocytes/embryos with mutation load below the threshold.
De Novo mtDNA Point Mutations
Counseling and Recurrence Risk
Besides being maternally inherited, mtDNA point mutation can also occur de novo in the affected individual, and this distinction makes a big difference for recurrence risk. If a de novo mutation is discovered in a child, this mutation is not expected to be present in his/her siblings. Due to the potential intra- and inter-tissue variability of mtDNA mutations, it can never be completely known for sure that the mother of the affected child does not carry any given mutation (i.e., a mutation load beneath the detection level, or the presence of a mtDNA mutation in any non-tested tissue, particularly the oocytes, would be impossible to exclude). However, proper analysis of multiple maternal tissues largely diminishes the residual risk of the mother having the mutation. Accordingly, for such de novo mtDNA point mutations, the recurrence risk is low, and the mutation is not expected to appear in a subsequent pregnancy. De novo mtDNA mutations are not rare events [1] Sallevelt et al in preparation, yet many such couples may be counseled incorrectly and given a high recurrence risk (erroneously), based on the high mutation load in the child instead of absence of the mutation in the mother.
Reproductive Testing Options
Given the low recurrence risk of apparently de novo mtDNA mutations, PND is feasible as reassurance Sallevelt et al in preparation. In four apparently de novo mtDNA disease cases based on the absence of the mutation in multiple maternal tissues, we have performed PND in a subsequent pregnancy. The mutation was not detected. In 9 of >100 reported cases describing apparently de novo mtDNA mutations, PND was performed in (a) subsequent pregnanc(y)(ies) with normal findings in the majority [35, 68–71], but recurrence in one family [49]. The latter might be the result of gonadal mosaicism, or of failed detection of very low mutation load in the mother’s lymphocytes and/or urinary epithelial cells due to the used sequencing method. This is currently being investigated further. PGD is, considering the burden of the treatment, not a favorable alternative in case of such a low recurrence risk.
mtDNA Rearrangements
Counseling and Recurrence Risk
Large, single mtDNA deletions are generally reported to occur sporadically, therefore having a low recurrence risk [72–74]. Indeed, the available data indicate that a clinically unaffected mother of an affected child has a negligible risk of another affected child [73]. Even for clinically affected mothers with an mtDNA deletion themselves, the risk of having clinically affected offspring is estimated to be low (1:24) [73]. mtDNA duplications are, like mtDNA point mutations, either maternally inherited or de novo and the same counseling aspects apply.
Reproductive Testing Options
PND seems the reproductive testing option of choice for de novo mtDNA rearrangements. Given the low recurrence risk even for women who carry an mtDNA deletion themselves, PND is the most feasible option in these cases, too. mtDNA duplications are, like mtDNA point mutations, either maternally inherited or de novo. For maternally inherited mtDNA duplications, the same considerations regarding reproductive testing options apply as for maternally inherited mtDNA point mutations.
Conclusion
Mitochondrial diseases are common metabolic disorders with potentially high morbidity and mortality. Generally, no treatment is available. Couples with a child affected by a mitochondrial disorder or a positive family history and a high risk of affected offspring may request prevention of transmission to a (future) child. Recurrence risks and the applicable reproductive testing options highly depend on the genetic etiology of the mitochondrial disease. For mitochondrial diseases due to nuclear gene defects, Mendelian segregation results in recurrence risks of 25 % or 50 %. Both PND and PGD are applicable, once the causative mutation has been identified. Recurrence risks particularly for mtDNA mutations should be determined on an individual basis, for example, taking into account the nature of the mutation and the mutation load in the mother. The risk for female carriers of mtDNA point mutations (such as the m.3243A>G mutation) of having affected offspring is often difficult to calculate, but it can be high. In those cases, PND is problematic mainly due to difficulties in predicting the phenotype with a given mutation load. PGD is currently the best reproductive testing option, although it should be regarded as a risk reduction strategy, rather than a method to exclude risk fully. Conversely, PGD is not the reproductive testing option of choice for apparently de novo mtDNA point mutations which have a low recurrence risk, making PND feasible for reassurance. The same is true for (large) mtDNA deletions which occur almost exclusively de novo. PND is also applicable for skewing mtDNA mutations, particularly when the mother has a low mutation load. The development of nuclear transfer technology would complete the portfolio of reproductive choices to prevent the transmission of mtDNA disease.
Acknowledgments
“Stichting Metakids”; referring neurologists and clinical geneticists; PND and PGD teams, Maastricht University Medical Centre+; Department of Obstetrics and Gynaecology, Maastricht University Medical Centre. The corresponding author had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure All authors are paid by the respective institutions according to his/her job contract. “Stichting Metakids” has funded research/work on mitochondrial diseases. The authors declare no conflicts of interest.
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