Gamete and Embryo Research Laboratory, Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, New Jersey, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: assisted reproduction/human oocytes/mtDNA fingerprinting/mitochondrial heteroplasmy/ooplasmic transplantation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this centre's ooplasmic transplantation programme, 12 clinical pregnancies were obtained after 28 attempts in 25 women. The clinical pregnancy rate was higher than expected (12 out of 28 cases), in a patient population with low fecundity. In some clinical cases significant improvement in embryonic development was seen after ooplasmic transfer. The basis for this work is the supposition that embryonic failure may be related to hitherto unknown cytoplasmic pathology. It is probable that a minimum threshold of ATP content is required for normal development, because cellular activities, e.g. chromosomal segregation, normal mitosis and physiological events during preimplantation development, require ATP. Furthermore, it is believed that there are significant differences in net ATP content in mature oocytes between patients (Van Blerkom et al., 1995).
Previously this laboratory has determined patterns of mitochondrial inheritance in embryos, amniocytes and fetal tissues after ooplasmic transplantation using the technique of mitochondrial DNA fingerprinting (Brenner et al., 2000). In addition to recipient maternal mtDNA, a small proportion of donor mtDNA has been detected in samples with the following frequencies: embryos (six out of 13), amniocytes (one out of four), placenta (two out of four) and fetal cord blood (two out of four). So far, there is no reason to consider the minimal proportion of detected hypervariable mtDNA heteroplasmy as harmful, particularly since it can occur spontaneously in normal individuals (Howell et al., 1992
; Bendall et al., 1995; Wilson et al., 1997
). Currently, there are 15 healthy children being monitored following the cytoplasmic transfer procedure. Because of the striking empirical success of this technique, the genomic aspects of ooplasmic transfer are being investigated, especially mitochondrial inheritance, as it relates to the presence and maintenance of mtDNA heteroplasmy among the offspring. Here, the question of heteroplasmy is addressed in two 1 year old children.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mtDNA fingerprinting
With IRB approval for clinical trials, 28 ooplasmic transplantation procedures in 25 couples were attempted. These couples participated in this study after previous conventional IVF attempts had failed due seemingly to persistent poor embryonic development. Each couple was counselled and consented to the known and unknown risks of this procedure and to the collection and study of any materials associated with this experimental technique. To determine the patterns of mitochondrial inheritance in the blood from two children after ooplasmic transplantation, mtDNA fingerprinting was performed as described previously (Brenner et al., 2000). The hypervariable region of the mitochondrial genome was amplified from the donor, recipient and the offspring's blood using the polymerase chain reaction (PCR) and the product was then DNA sequenced to determine the mitochondrial fingerprint (Brenner et al., 2000
). The mitochondrial fingerprints (DNA chromatographs) of blood samples obtained from two children, aged 9 months and 14 months after paediatric evaluations, were then compared with the donor and recipient mtDNA fingerprints.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Confocal analysis of preimplantation embryos showed distribution of mitochondria to a population of the cells and fragments. Similarly, the mtDNA heteroplasmy detected in the amniocytes, placental and fetal cord blood samples (Brenner et al., 2000) suggests that differences in the distribution of the donor mitochondria to fetal tissues may vary. The analysis of the distribution of the donor mitochondria in both embryonic and extra-embryonic tissues will require further investigation. Furthermore, the determination of whether donor mitochondria are actively segregated or randomly segregated within developing preimplantation embryos, embryonic and extra-embryonic tissues may even shed some light on the mitochondrial bottleneck theory (Hauswirth and Laipis, 1982
).
mtDNA fingerprinting analysis performed on blood samples from 1 year old children following ooplasmic transfer detected mitochondrial polymorphisms in which both alleles were present in the hypervariable region of the mitochondrial genome. It has been previously estimated (Nickerson et al., 1997) that the minor allele must be present in at least 30% of the sample to be detected on the DNA chromatograph and therefore the donor allele must be highly represented in the blood samples of the offspring. Furthermore, highly sensitive molecular analysis using allele specificPCR (ASPCR) or molecular beacons will be necessary to determine the frequency of donor and recipient mtDNA heteroplasmy in the blood samples or buccal smears derived from babies of the ooplasmic transplantation procedures.
These are the first reported cases of germline mtDNA genetic modification which have led to the inheritance of two mtDNA populations in the children resulting from ooplasmic transplantation. These mtDNA fingerprints demonstrate that the transferred mitochondria can be replicated and maintained in the offspring, therefore being a genetic modification without potentially altering mitochondrial function. Furthermore, there has been no alteration of nuclear DNA inheritance in the fetal cord blood from these babies (Brenner et al., 2000).
Presently, there is no reason to consider the minimal proportion of detected donor mitochondria observed in the offspring as harmful, since it is known to occur naturally in normal individuals (Ivanov et al., 1996). Also, there is an important discrepancy between benign heteroplasmy after mixing two potentially normal populations of mitochondria, as reported here, compared to pathological heteroplasmy which occurs during the ageing process or in patients with mitochondrial disease. We consider that the perceived risk to offspring from cytoplasmic transfer is minimal, and therefore we continue to perform these clinical procedures, albeit as an experimental protocol. However, the regulation of the donor's mitochondrial population and how it is maintained by the recipient's nuclear genome must be continually evaluated in the offspring from cytoplasmic transfer.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brenner, C.A., Barritt, J.A., Willadsen, S. and Cohen J. (2000) Mitochondrial DNA heteroplasmy after human ooplasmic transplantation. Fertil. Steril., 74, 573578.[ISI][Medline]
Cohen, J., Scott, R., Schimmel, T. et al. (1997) Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet, 350, 186187.[ISI][Medline]
Cohen, J., Scott, R., Alikani, M. et al. (1998) Ooplasmic transfer in mature human oocytes. Mol. Hum. Reprod., 4, 269280.[Abstract]
Hauswirth, W.W. and Laipis, P.J. (1982) Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc. Natl Acad. Sci. USA, 79, 46864690.[Abstract]
Howell, N., Halvorson, S., Kubacka, I. et al. (1992) Mitochondrial gene segregation in mammals: is the bottleneck always narrow? Hum. Genet., 90, 117120.[ISI][Medline]
Ivanov, P.L., Wadhams, M.J., Roby, R.K. et al. (1996) Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nature Genet., 4, 417420.
Nickerson, D.A., Tobe, V.O. and Taylor, S.L. (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res., 25, 27452751.
Perez, G.I., Trbovich, A.M., Gosden, R.G. and Tilly, J.L. (2000) Mitochondria and the death of oocytes. Nature, 403, 500501.[ISI][Medline]
Van Blerkom, J., Davis, P.W. and John, L. (1995) ATP content and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum. Reprod., 10, 415424.[Abstract]
Van Blerkom, J., Sinclair, J. and Davis, P. (1998) Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy. Hum. Reprod., 13, 28572868.
Wilson, M.R., Polanskey, D., Replogle, J. et al. (1997) A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes. Hum. Genet., 100, 167171.[ISI][Medline]
Submitted on September 15, 2000; accepted on December 8, 2000.