Mitochondrial DNA deletion in human oocytes and embryos

Molecular Human Reproduction vol.4 no.9 pp. 887–892, 1998

Mitochondrial DNA deletion in human oocytes and embryos

Carol A.Brenner1,3, Yvonne M.Wolny1, Jason A.Barritt2, Dennis W.Matt2, Santiago Munne´1 and Jacques Cohen1 1The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, Gamete and Embryo Laboratory, Livingston, New Jersey, and 2Medical College of Virginia, Virginia Commonwealth University, Department of Obstetrics and Gynecology, Richmond, VA, USA 3To

whom correspondence should be addressed

Mitochondrial DNA (mtDNA) deletions are present in both human oocytes and embryos. It has been found that these tissues contain a mtDNA mutation which is present in high amounts in patients with Kearns– Sayre syndrome (KSS) and progressive external ophthalmoplegia. In the present study, the frequency of this KSS deletion was investigated in human oocytes and embryos. Using a nested primer polymerase chain chian reaction (PCR) strategy, the frequency of the KSS deletion in 74 human oocytes and 137 embryos was found to be 32.8 and 8.0% respectively. Using a ‘long PCR–short PCR’ nested primer strategy, the frequency of the KSS deletion in 181 human oocytes and 104 embryos was found to be 47.0 and 20.2% respectively. There was no statistical correlation between the age of the patients at the time of oocyte retrieval and the presence of the deleted molecules. There was a statistical difference between the presence of the deleted molecules in oocytes versus embryos using either technique (P < 0.0001). The relevance of these findings to the accumulation of low levels of deleted mtDNA in both oocytes and embryos is discussed in this study. Key words: human embryos/human oocytes/KSS deletion/mitochondrial DNA/reproductive ageing

Introduction Mutations in mitochondrial DNA (mtDNA) are responsible for certain neuromuscular syndromes, the most common being Kearns–Sayre syndrome (KSS) or progressive external ophthalmoplegia (PEO) (Holt et al., 1988; Schon et al., 1994; Wallace et al., 1997). A large, 4977 bp mtDNA deletion occurs in a ‘hot spot’ region (Holt et al., 1988) involving two 13 bp repeats beginning at positions 8470 and 13447. The frequency of this mtDNA deletion in affected individuals was 30–90% (Cortopassi and Arnheim, 1990; Schon et al., 1994; Zeviani and Antozzi, 1997). The same mutation has been found to accumulate in somatic tissues during the normal life span, but at a much lower frequency of 0.1% (Cortopassi and Arnheim, 1990; Arnheim and Cortopassi, 1992; Simonetti et al., 1992; Lee et al., 1994). Interestingly, fetal tissue does not contain any mtDNA mutations, as opposed to adult tissue; suggesting a close relationship between mtDNA mutations and ageing (Arnheim and Cortopassi, 1992). Furthermore, slow and nondividing cells, including neurons, myocytes, and oocytes, appear to display a higher percentage of mitochondrial mutations than rapidly dividing ones, possibly because of prolonged exposure to DNA mutagens. mtDNA is in close proximity to highly mutagenic reactive oxygen species (ROS), which are generated within mitochondria; this is believed to cause DNA damage, in turn leading to bioenergetic deficit, cell death, and reproductive senescence (Ozawa et al., 1995). Hauswirth and Laipis (1982) postulated that deleted mtDNA cannot be transferred from the oocyte to the implanted embryo, because of the postulated presence of a ‘bottleneck’, limiting © European Society for Human Reproduction and Embryology

the number of transmitted mtDNA. This theory argues that after oogenesis and before embryogenesis, there is a reduction in the total number of mtDNA and therefore an elimination of mutated mtDNA. According to this hypothesis, the expected frequency and percentage of mutated mtDNA in human embryos and blastocysts should be low or possibly absent, when compared with oocytes. Howell et al. (1992) suggested that the mtDNA segregation occurs during oogenesis and embryogenesis in a slow mode. The slow mode implies that there is no significant segregation of mtDNA among family members or between generations as evaluated in a family with Leber hereditary optic neuropathy. A recent report (Cohen et al., 1997) on the birth of a child following transfer of anucleate donor ooplasm from younger women into recipient eggs raises questions about the possibility of transmission of mutated mtDNA. The zygote becomes the recipient of three cytoplasms, two from the oocytes and one from the sperm, that may potentially harbour deleterious mutations. In this cytoplasmic donation case, mitochondrial fingerprinting analysis proved that there was no detectable donor mtDNA in amniocytes (Cohen et al., 1998). Also, no transmission of mitochondrial DNA to the recipient offspring occurred. Experiments are in progress to evaluate whether mtDNA from anucleate donor ooplasm transferred into the recipient oocyte can be maintained during embryogenesis and in the newborn (J.A.Barritt, J.Cohen and C.A.Brenner, unpublished results). The purpose of this study was to determine the frequency of the KSS deletion in human oocytes and embryos and its relationship to reproductive ageing. 887

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Table I. Polymerase chain reaction (PCR) primers to detect the Kearns–Sayre syndrome (KSS) mitochondrial DNA (mtDNA) deletion Primer Name

Origination

Sequence

MT-1 (8468) MT-2 (13176) MT-3 (13551) MT-4 (13707) 5835 (5818) 16065 (16097)

59 59 39 39 59 39

59 59 59 59 59 59

CTT AGG AGA CTG GAA ACG

TGA CGC GTA CGA TCT GAA

AGT TAT ATA ATA AGA TTC

AGG CAC GAT GGC CTC ATA

GCC CAC AGG TTC GGA GCG

CGT TCT GCT CGG GCT GTT

ATT TGT CAG CTG GGT GTT

TAC 39 TCGC 39 GCG 39 CC 39 AAA AAG 39 GAT GGG TGA GTC 39

Table II. Frequency of deleted mitochondrial DNA (mtDNA) in human oocytes and embryos by nested polymerase chain reaction (PCR) (technique no. 1)

Oocytes Embryos

No. analysed

No. amplified

KSS deleted mtDNA

Non-deleted mtDNA Frequency (%)

76 139

73 137

24 11

49 126

32.8a 8.0a

significant difference (P , 0.0001) by χ2 analysis. A comparison of the number of samples analysed and the number amplified indicates the amplification efficiency.

aStatistically

Table III. Frequency of Kearns–Sayre syndrome (KSS) deleted mitochondrial DNA (mtDNA) in human oocytes and embryos by long polymerase chain reaction PCR–short PCR (technique no. 2)

Oocytes Embryos

No. analysed No. amplified

KSS deleted mtDNA

Non-deleted mtDNA

Frequency of KSS deleted mtDNA (%)

220 121

85 21

96 83

46.96a 20.19a

181 104

significant difference (P , 0.0001) by χ2 analysis. A comparison of the number of samples analysed and the number amplified indicates the amplification efficiency.

aStatistically

Materials and methods Collection of human oocytes and embryos In-vitro matured and unfertilized oocytes were obtained from consenting couples. Preimplantation embryos were donated when considered unsuitable for embryo replacement or cryopreservation or in cases where cryopreservation was not permitted. All research and use of patient material was performed in accordance with approved IRB protocols of Saint Barnabas Medical Center (SBMC) and the Medical College of Virginia (MCV). Ovarian stimulation was performed by down-regulation and gonadotrophin substitution. Donated oocytes and embryos were washed three times in sterile phosphate-buffered saline (PBS) in sterile plastic dishes, and then transferred to a 0.2 ml tube in 1–3 µl volume. Two different polymerase chain reaction (PCR) techniques were developed to evaluate the KSS deletion in oocytes and embryos. For technique no. 1, oocytes and embryos were obtained from 49 patients collected at SBMC; 73 unfertilized metaphase II (MII) oocytes from 17 patients and 137 discarded day 3 embryos from 35 patients. For technique no. 2, oocytes were collected at MCV and embryos were collected at SBMC; 181 oocytes from 51 patients and 104 embryos from 33 patients (see Tables II and III). The immature oocytes, oocytes that failed to fertilize and the preimplantation embryos used in this study were considered unsuitable for embryo replacement because they were considered suboptimal, as defined by our oocyte and embryo classification system. Embryo classification Embryos were classified by their morphology (Munne´ et al., 1995). Arrested embryos had not developed beyond the 4-cell stage on the

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third day of development and had not cleaved during a 24 h period. Slow embryos were those that had not reached the 6-cell stage on day 3 of development. Human preimplantation embryos often appear with one or more of their blastomeres fragmented. Human embryos developing in vitro not only exhibit different degrees of fragmentation, they often fragment in specific patterns. We have recently defined five such patterns of fragmentation (Warner et al., 1998). Multiple nucleii or multinucleation in at least one cell is common in human preimplantation embryos. The occurrence of multinucleated blastomeres (MNB) occurs any time between the first cleavage division and the blastocyst stage (Munne´ and Cohen, 1993).

DNA purification (technique no. 1) Gene Releaser (Bio Ventures Inc, Murfreesboro, TN, USA) (5 µl) was added to a tube containing single oocytes or embryos. The tubes were microwaved for 5 min (900 V) to release DNA. The sample was frozen at –70°C. Prior to PCR amplification the sample was heated for 5 min at 70°C. The DNA from TC613 cell line was isolated using a Puregene DNA Isolation Kit (Gentra Systems Inc, Minneapolis, MI, USA). The TC613 cell line is a fibroblast cell line, obtained from a KSS patient and was a gift from Dr Nancy Kennaway, Oregon, USA. PCR amplification (technique no. 1) The gene-specific oligonucleotide primers used in this study were synthesized by Genosys Biotechnologies Inc (The Woodlands, TX, USA). In order to amplify the control mtDNA and the deletions simultaneously, the DNA was divided into two aliquots prior to PCR

mt DNA deletion in oocytes and embryos

Figure 1. Nested polymerase chain reaction (PCR) strategy applied to detect the Kearns–Sayre syndrome (KSS) mitochondrial DNA (mtDNA) deletion. Primers MT-1 and MT-4 were flanking the mitochondrial ‘hot spot’ in order to identify the 4977 bp deletion. PCR primers MT-2, MT-3 were used as a control for the presence of mtDNA and were outside the deleted region. amplification. Each aliquot of DNA was amplified using a 9600 Perkin Elmer system in a 50 µl reaction volume containing a final concentration of 1.5 mM MgCl2, 1 mM dNTP mix, 0.5 µM each gene-specific primer, and 1.25 IU Taq polymerase (Quiagen, Santa Clarita Inc, CA, USA) using the following amplification profile: 1 cycle of 96°C 3 min; 40 cycles 94°C, 20 s, 55°C, 20 s, 72°C, 20 s, 1 cycle, 72°C 7 min; hold at 4°C. For the nested PCR, 2.5 µl of the first PCR reaction product served as a template. The procedure was carried out using the same PCR conditions as before. The evaluation of PCR results was performed by agarose gel electrophoresis; 10 µl of nested PCR product was separated on 2% agarose gel and stained with ethidium bromide. The positive control for the KSS deletion was DNA isolated from a TC613 cell-line derived from a patient with KSS syndrome which was amplified using the same conditions as for oocytes and embryos mtDNA. Substitution of DNA template with a dH2O served as a negative control. PCR products were confirmed by DNA sequencing.

Nested PCR Strategy (technique no. 1) In order to detect the KSS deletion, a nested PCR strategy was applied. The sequence of PCR primers used in this study was: sense MT-1 and MT-2 and anti-sense MT-3, MT-4 is shown in Table I. The MT-1 primer flanked the KSS deletion (8468 bp) on the 59 side, while MT-4 (13707 bp), MT-3 (13551 bp) flanked on the 39 side of the deletion (Figure 1). The MT-3 primer was used as an internal primer for the nested PCR reaction. If the KSS mtDNA deletion was present, the nested PCR product was 349 bp long. In the absence of the deletion, there was no DNA amplification because the PCR primers were too widely separated to amplify the 5 kb product. The undeleted mtDNA was amplified using primers MT-2 (13176 bp), MT-4 followed by MT-2, MT-3 and resulted in products 550 and 398 bp respectively. ‘Long PCR–short PCR’ strategy (technique no. 2) In order to verify the results of the nested PCR (technique no. 1) a second strategy to amplify the KSS mtDNA deletion was designed. This technique uses a long PCR followed by a short PCR reaction with nested primers. The long PCR technique uses a proof-reading polymerase and is able to amplify large regions of DNA. In this study two primers (5835 and 16065, see Table I for sequences) separated by 10.2 kb of the mtDNA are used in the long PCR reaction. This single long PCR reaction amplifies all normal and deletion containing mtDNA in the sample, so the sample is not split

as in the PCR reactions for technique no. 1. The two second-round PCR reactions for normal mtDNA and for the KSS deletion use 1 µl of the long PCR reaction product as template; they are amplified following the same procedure as for the second-round nested PCR reactions in technique no. 1, as previously discussed.

Mitochondrial DNA preparation (technique no. 2) All samples were frozen in 16 µl of sterile water to –70°C to cause cell lysis. Samples were thawed at room temperature for 10 min followed by the addition of 1.7 µg of Proteinase K (Promega, Madison, WI, USA), 10 µl sterile water and 10 µl eLongase 53 PCR buffer with 2.0 mM Mg (Life Technologies Inc, Gaithersburg, MD, USA). Samples were incubated at 37°C for 30 min, 50°C for 30 min, and 95°C for 10 min. ‘Long PCR–short PCR’ amplification (technique no. 2) For the 50 µl long PCR reaction 1 mM dNTP mix, 250 pM each specific primer (5835 and 16065), 1 IU eLongase polymerase (Life Technologies Inc) and 11 µl sterile water are added at 80°C for a hot start to the long PCR reaction. The following amplification profile was used in both a Perkin-Elmer 9600 and a MJ Research PTC-100 with no difference in amplification seen: 1 cycle of 94°C for 1 min; 35 cycles of 94°C, 20 s, 62°C, 20 s, 68°C, 12 min; 1 cycle of 72°C for 10 min; and a final hold at 4°C. Positive and negative control reactions were run in concert with each sample amplification. The positive control was from the eLongase Amplification Kit (LifeTechnologies) which amplifies a 20 kb sequence using the primers and template provided. This positive control allowed for determination of the proper functioning of the PCR machine and the correct addition of functional reagents to the reactions. A negative control containing all the reagents for sample amplification but containing only sterile water instead of a sample was run with each amplification reaction. The negative control never produced any mtDNA amplification. The evaluation of PCR results was performed by agarose gel electrophoresis using 10 µl of long PCR product separated on 0.8% agarose gel stained with ethidium bromide. The second-round short PCR reactions for both normal mtDNA and the KSS deletion were carried out using the same primers and conditions as for the second-round amplifications in the nested PCR technique no. 1. For normal mtDNA primers MT-2 and MT-4 were used, and for the KSS deletion primers MT-1 and MT-3 were used. Only samples that contained a normal mtDNA product (550 bp) with MT-2 and MT-4 were included in this study.

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Figure 2. Mitochondrial DNA (mtDNA) Kearns–Sayre syndrome (KSS) deletion and control mtDNA present in human oocytes. Nested PCR products were separated on 2% agarose gel, stained with ethidium bromide. Lane M: 1 kb DNA ladder. Lane 1, 7, 8 and 11 (left side) demonstrated the KSS mtDNA deletion amplified with primers MT-1, MT-4 followed by MT-1, MT-3, showing a band 349 bp long. The different intensity of polymerase chain reaction (PCR) products indicates a different copy number of deleted mtDNA in each sample. Control, undeleted mtDNA was amplified with MT-2, MT-4 and MT-2, MT-3. The nested PCR product was 398 bp long.

Statistical analysis The statistical correlation between the incidence of the KSS mutation and the age of the patient’s oocytes and embryos was investigated by carrying out a logistic regression analysis, the dependent variable being the logistic transform of the proportions of mutated oocytes/ embryos, and the age being the single explanatory variable. The algorithm Genstat was used to carry out the statistical calculations. χ2 analysis of the numbers of oocytes verses the numbers of embryos that harbour the KSS deletion was performed using Sigma Stat.

Results KSS mitochondrial DNA deletion in human oocytes Using technique no. 1, a total of 73 oocytes from 17 patients were analysed, of which 24 (32.8%) showed the deletion (Table II). Using technique no. 2, a total of 181 oocytes from 51 patients were analysed, of which 85 (47.0%) showed the KSS deletion (Table III). The amplification efficiency was 96% using technique no. 1, compared with 82.2% using technique no. 2. As shown in Figure 2, mtDNA from oocytes in lanes 1, 7, 8 and 11 indicated the deletion. The intensity of amplified PCR product varied considerably, probably caused by different copy numbers of deleted mtDNA between the oocytes analysed. DNA obtained from a TC613 fibroblast cell line from one patient with KSS syndrome served as a positive control and showed the 5 kb deletion in each PCR amplification. In each PCR reaction, template DNA was substituted with H2O which served as a blank control, to rule out the possibility that contamination caused the PCR amplification. No contamination was found with any DNA during each PCR amplification. Mitochondrial DNA deletions in human embryos Using technique no. 1, a total of 137 embryos from 35 patients were analysed, of which 11 (8.0%) showed the KSS deletion (Table II). The KSS deletion in the embryos was evaluated using the same strategy as applied to the oocytes. Analysis of the embryos’ mtDNA deletion is summarized in Figure 3 and Table II. Figure 3 shows the KSS mtDNA deletion in the TC613 cell-line obtained from a KSS patient; the nested PCR product was 349 bp long. DNA from embryos in lanes 3 and 4 showed the deletion and a substantial difference in band intensity, most likely reflecting a difference in mtDNA copy number. Occasionally, there is a larger amplification product in the control mtDNA (Figure 2; lanes 1, 7 and 12). This PCR 890

product has been DNA sequenced and determined to be an artefact. In each PCR reaction, a blank control was applied and did not show any DNA contamination. Only samples which showed positive amplification using MT-2, MT-4 followed by MT-2, MT-3 were analysed; two out of 139 embryos were negative for the undeleted mtDNA; indicating no mtDNA in the analysed embryos. Using technique no. 2, a total of 104 embryos from 33 patients were analysed, of which 21 (20.2%) showed the KSS deletion (Table III). The amplification efficiency was 98.6% using technique no. 1, compared with 86.0% using technique no. 2. Long PCR is always less efficient because large regions of DNA are being amplified. In this study, primers separated by 10.2 kb of the mtDNA are used in the long PCR reaction.

Relationship of mtDNA deletion between human oocytes and embryos and patient age No correlation was found between (i) the mitochondrial deletion in oocytes (b 5 0.018 6 0.069) and embryos (b 5 – 0.107 6 0.094) and (ii) patient age as calculated using the Genstat algorithm; perhaps due to the small number of cases in this study. The average age of patients was 36.02 years, and the average age of patients whose gametes demonstrated the KSS mitochondrial deletion was 34.50 years. Ratio of oocytes versus embryos that harbour the KSS deletion χ2 analysis of the results obtained using the nested PCR technique no. 1 (Table II) showed a statistically significant difference (P , 0.0001) between the number of oocytes harbouring the KSS deletion (24/73) and the number of embryos harbouring the KSS deletion (11/137). χ2 analysis of the results obtained using the ‘long PCR–short PCR’ technique no. 2 (Table III) showed a statistically significant difference (P , 0.0001) between the number of oocytes harbouring the KSS deletion (85/181) and the number of embryos harbouring the KSS deletion (33/104).

Discussion The data obtained in this study indicates that both human oocytes and embryos harbour a KSS deletion in their mtDNA, but the percentage of this mutation is statistically significantly reduced after fertilization (Tables II and III). Using technique

mt DNA deletion in oocytes and embryos

Figure 3. Representation of the Kearns–Sayre syndrome (KSS) mitochondrial DNA (mtDNA) deletion, and control undeleted mtDNA in human embryos. The polymerase chain reaction (PCR) products were run on 2% agarose gel. The KSS deletion and control mtDNA were amplified as in human oocytes. Lane M: 100 bp DNA ladder. Lanes 1 and 2: KSS mtDNA deletion identified in DNA isolated from a TC613 fibroblast cell lines obtained from a KSS patient. Lanes 3 and 4: (left side) represent 349 bp long mtDNA deletion in human embryos. The control 398 bp mtDNA was identified in each sample (right side).

no. 1, the frequency of the KSS deleted mtDNA was 32.8% in oocytes and 8.0% in the embryos, compared with 47.0% in oocytes and 20.2% in embryos using technique no. 2. Although the two techniques showed different percentages of oocytes and embryos harbouring the KSS deletion, technique no. 2 may have found higher percentages of deleted mtDNA because the sample was not split prior to analysis. Furthermore, there were other differences such as a different selection of gametes, and a different method of lysing the cells prior to PCR amplification. There was no correlation between the mtDNA deletion in human gametes and patient age, suggesting that this deletion is not a marker for reproductive senescence. Using technique no. 1, our results show a similar frequency of mtDNA deletions in oocytes (33%) as reported by Chen et al. (1995) (n 5 104, 49%) and Barritt et al. (1997) (n 5 96, 35%). Neither group detected any correlation between the mtDNA deletion and patient age. On the other hand, Keefe et al. (1995) showed a KSS mtDNA deletion in 43% of the 50 oocytes analysed, but found a significant association between patient age and mtDNA mutation. Kitagawa et al. (1993), reported that in ovarian tissue the mtDNA deletion accumulates rapidly at the menopausal period and is related to the age of the reproductive ovary. Since we did not find any correlation between the KSS deletion in gametes and patient age, the mutation cannot be used as a potential cytoplasmic marker for maternal reproductive senescence. However, since the ageing process is associated with an excessive amount of oxidative damage, which may be correlated with the accumulation of deleted mtDNA in several tissues, oocytes from older patients may have reduced respiratory function (Arnheim and Cortopassi, 1992). Furthermore, we have begun to look at other rearrangements of mtDNA as well as point mutations of mtDNA in oocytes and embryos (J.A.Barrit, C.A.Brenner, J.Cohen and D.W.Matt, unpublished results). Apart from the low numbers of patients in our study, the lack of finding could be because our in-vitro fertilization (IVF) patients are preselected for parameters of reproductive senescence, such an elevated day 3 concentration of follicle stimulating hormone (FSH) or low response to gonadotrophins. Thus far, there have been no reports about the frequency of mtDNA deletions in human oocytes compared with embryos. This study reports the frequency of deleted mtDNA in oocytes and embryos and demonstrates that the shift in the mutation decreases with embryo development. Although total mitochon-

drial DNA content remains constant between the mature egg and the blastocyst stage in mammals (Ebert et al., 1988; Meirelles et al., 1998), it is not known what happens to mitochondrial mutations like KSS deletions during human embryogenesis. Irrespective of which PCR technique was used, there was a decrease in the percentage of mtDNA deletions in embryos compared with oocytes. It has always been suspected that even if there are mtDNA mutations present in human oocytes, a mitochondrial ‘bottleneck’ would filter out mutated mtDNA, as mentioned above. Consequently, mitochondrial integrity is supposed to be preserved in each generation. Since we were still able to identify mtDNA deletions in embryos, although at a much lower frequency than in oocytes, our study did not entirely confirm the ‘bottleneck’ theory. But the discrepancy and decreasing tendency of a mtDNA deletion between oocytes and embryos could indicate that sporadic mutations accumulate in oocytes during oogenesis and are eliminated by an unknown, perhaps nuclear mechanism. Possibly, a large percentage of these oocytes with this mutation simply die and do not develop into embryos. In the latter case, it would be interesting to assess the occurrence and copy number of deleted KSS mtDNA and other mutations during the development of embryos, blastocysts, amniocytes, and fetal tissue, in the same individual. In the future, we do expect to be able to evaluate mtDNA deletions in offspring. A recent report describes a successful birth following transfer of anucleate donor oocyte cytoplasm into recipient eggs (Cohen et al., 1998). This raises the question whether mutated mtDNA can be transmitted to the newborn, since the physiology of the oocyte is now affected by three genomes (Brenner et al., 1997). Since mitochondrial DNA is maternally inherited, it is not clear how multiple mitochondrial mutations associated with oligoasthenozoospermia may be transmitted to the offspring (Lestienne et al., 1997). Mitochondrial disease occurs only if there is a high ratio of heteroplastic, wild-type:mutated (30– 90 versus 0.1%) mtDNA; which further supports the belief that transfer of a low copy number of mutated mtDNA into a recipient cytoplasm would not be sufficient to cause any disease (Lapis et al., 1988; Hayashi et al., 1991). Furthermore, the nucleus may be the regulator of mtDNA inheritance and transmission. Electrofusion of a heteroplastic cytoplast into a recipient 1-cell mouse embryo resulted in either the complete absence of donor mtDNA in the offspring; or in transmission 891

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of 29.6% of donor mtDNA to the offspring (Kaneda et al., 1995). A similar mechanism may be responsible for the elimination of paternal mtDNA, post-fertilization. Elimination of paternal mtDNA in intra-, but not inter-specific, hybrids during early embryogenesis implies a species-specific mechanism involved in recognition and destruction of sperm mtDNA, as was demonstrated in mice (Kaneda et al., 1995). Our work continues on the KSS mtDNA deletion in similar patients’ oocytes, embryos, amniocytes and newborn tissue; but in summary, the fact that there is a substantial reduction of the KSS mtDNA deletion in human embryos indicates a very remote probability of transmission of mutated mtDNA to the newborn following aggressive micromanipulation.

Acknowledgements The authors gratefully acknowledge the efforts of the team of embryologists at the Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center; Giles Tomkin for editorial assistance; and Doctors Richard Scott and Paul Bergh for their support of this study. The authors also acknowledge the embryology staff at the Medical College of Virginia. Our thanks to Dr D.E.Walters for statistical calculations.

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