Quantification of human ooplasmic mitochondria.

It is likely that there is an association between the fitness of mitochondria and their ability to support normal cellular function. Oocytes are greatly enriched in the number of mitochondria as they support essential developmental processes such as oocyte maturation and embryonic development, while their replication is deferred until gastrulation. The mitochondrial DNA (mtDNA) copy number in 87 human oocytes from 29 patients was evaluated after DNA extraction and real-time quantitative polymerase chain reaction (PCR). The average mtDNA copy number was 795,000 (+/- 243,000) in metaphase II oocytes. mtDNA content varied considerably between oocytes, even within the same patient. No relationship was found between mtDNA copy number and maternal age. The findings suggest that mtDNA replication is fully accomplished by the germinal vesicle stage in the fully developed oocyte.

Mitochondria in human offspring derived from ooplasmic transplantation.

Ooplasmic transfer from fertile donor oocytes into potentially compromised recipient patient oocytes has led to the birth of nearly 30 babies worldwide. Cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial (mt) heteroplasmy (both recipient and donor mtDNA) in offspring. This investigation traced the mitochondrial donor population both during the ooplasmic transfer technique and in the bloods of two 1 year old children using mtDNA fingerprinting. Donor ooplasm stained for active mitochondria was transferred into recipient ooplasm and the mitochondria were visualized by confocal microscopy after the microinjection procedure and fertilization. Heteroplasmy was found in the blood from each of the children. This report is the first case of human germline genetic modification resulting in normal healthy children.

Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR.

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.

Spontaneous and artificial changes in human ooplasmic mitochondria.

Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Because of the enigma of mitochondrial heteroplasmy, the mixing of populations of oocyte cytoplasm has provoked considerable debate. We are currently investigating oocyte mitochondrial (mt) DNA mutations and the effects of ooplasmic transplantation on mitochondrial inheritance and mitochondrial functionality. Ageing human oocytes could accumulate mtDNA deletions, which might lead to detrimental development. Elimination of abnormal, rearranged mtDNA, such that the offspring inherit only normal mitochondria, is postulated to occur by a mtDNA 'bottleneck'. Among compromised human oocytes (n = 74) and early embryos (n = 137), investigations have shown the occurrence of deltamtDNA4977, the so-called common deletion, to be 33% among oocytes and 8% among embryos. Using a nested polymerase chain reaction (PCR) strategy of long followed by short PCR, another 23 novel mtDNA rearrangements were found: various rearrangements were present in 51% of the oocytes (n = 295) and 32% of early embryos (n = 197). The difference in the percentage of mtDNA rearrangements between oocytes and embryos was significant (P < 0.0001) and implies that there could be a process of selection as fertilized oocytes become embryos. There was no significant relationship between the percentage of human oocytes or embryos that contained mtDNA rearrangements and age. The first series of ooplasmic transfers have been performed in women with repeated implantation failure associated with slow and morphologically abnormal development of their embryos. In a total of 23 attempts in 21 women, eight healthy babies have been born and other pregnancies are ongoing. By examining the donor and recipient blood samples it is possible to distinguish differences in their mtDNA fingerprint. A small proportion of donor mitochondrial DNA was 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). Ooplasmic transfer can thus result in sustained mtDNA heteroplasmy representing both the donor and recipient.

Mitochondrial DNA heteroplasmy after human ooplasmic transplantation.

OBJECTIVE: To determine the patterns of mitochondrial inheritance in embryos, fetuses, and infants after ooplasmic transplantation using the technique of mitochondrial DNA (mtDNA) fingerprinting. DESIGN: Prospective clinical study. SETTING: The IVF program at Saint Barnabas Medical Center, a nonprofit community hospital. PATIENT(S): In a total of 23 cases with recurrent implantation failure after IVF ooplasmic transplantation was performed. Thirteen embryos from two patients and amniotic cells from four patients were investigated for heteroplasmy. Placenta and fetal cord blood cells from four newborn babies/infants were also investigated. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): mtDNA fingerprinting, polymerase chain reaction, and DNA sequencing analysis. RESULT(S): In addition to the recipient maternal mitochondrial DNA, a small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (n = 6/13), amniocytes (n = 1/4), placenta (n = 2/4), and fetal cord blood (n = 2/4). Fingerprinting showed that nuclear DNA was not inherited from the donor in placenta or fetal cord blood of the babies. CONCLUSION(S): Ooplasmic transfer can result in sustained mtDNA heteroplasmy representing both donor and recipient. This was shown by mtDNA fingerprinting of embryos, amniocytes, fetal placenta, and cord blood. These results show that the donor-derived mitochondrial population persists after ooplasmic transfer and may be replicated during fetal development.

Mitochondrial DNA point mutation in human oocytes is associated with maternal age.

Mitochondrial DNA (mtDNA) point mutations are known to accumulate in an age-dependent fashion in somatic tissues. This study investigated whether a point mutation (T414G) in the mtDNA control region was present in oocytes from women of advanced age. In all, 66 non-viable discarded human oocytes were analysed for the presence of a T414G transversion mutation. DNA sequence analysis confirmed the presence of this mutation in one oocyte from 11 patients between the ages of 26 and 36 years (n = 23), compared to 17 oocytes from 10 patients between the ages of 37 and 42 years (n = 43). The younger group exhibited this mtDNA point mutation in only 4.4% of oocytes compared to 39.5% from the older group (P < 0.01). Therefore, single human oocytes contain the mtDNA T414G transversion point mutation that accumulates in an age-dependent manner. The potential significance of this point mutation may be its association with reproductive senescence. Furthermore, since this mutation exists in the control region of the mtDNA it may affect the regulation of mtDNA transcription and replication during oocyte and post-embryonic development.

Mitochondrial DNA rearrangements in human oocytes and embryos.

Human mitochondrial DNA (mtDNA) rearrangements, including more than 150 deletions and insertions, accumulate with age and are responsible for certain neuromuscular diseases. Human oocytes, arrested for up to 50 years, may express certain mtDNA rearrangements possibly affecting function. Investigations have previously shown a single mtDNA rearrangement (dmtDNA(4977)) in human oocytes. Sequencing of other rearrangements and their correlation with maternal age have not been performed in human oocytes or embryos. Here we use a nested PCR strategy of long followed by short polymerase chain reaction (PCR) that amplifies two-thirds of the mitochondrial genome. mtDNA rearrangements were detected in 50.5% of the oocytes (n = 295) and 32.5% of the embryos (n = 197). This represents a significant difference in the percentage of mtDNA rearrangements between oocytes and embryos (P < 0.0001). Twenty-three novel mtDNA rearrangements with deletions, insertions and duplications were found. There was no significant age-related increase in the percentage of human oocytes or embryos that contained mtDNA rearrangements. Significant reductions in the number of oocytes containing mtDNA rearrangements occurred as oocyte development progressed from germinal vesicle to the mature metaphase II oocyte (P < 0.05). These findings are discussed as they relate to mitochondria, mtDNA, and ATP production in human oocytes and embryos.

Mitochondrial DNA deletion in human oocytes and embryos.

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.

Psychosocial aspects of head and neck cancer surgery.

Thirty-six patients who underwent major surgical excision and reconstruction for head and neck cancer were subjected to a psychosocial assessment in hospital and, later, in their home environments. The information gained from the survey is discussed, with particular reference to those factors which can be used by the head and neck surgical team to improve the quality of the patients' existence.