Carol A. Brenner

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.

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence.

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6‐fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10‐fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/103 cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line. J. Cell. Physiol.

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.

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.

Antiphospholipid antibodies and pregnancy rates and outcome in in vitro fertilization patients.

OBJECTIVE To determine the relationship between antiphospholipid antibodies and pregnancy rates (PRs) and outcome among IVF patients. DESIGN Prospective collection of all serum samples with assays for immunoglobulin G (IgG), IgA, and IgM antibodies for anticardiolipin, antiphosphatidyl serine, antiphosphatidyl ethanolamine, antiphosphatidyl choline, antiphosphatidyl inositol, antiphosphatidyl glycerol, and antiphosphatidic acid being done following completion of all treatment cycles. SETTING A tertiary care teaching hospital. PATIENT(S) Seven hundred ninety-three patients attempting to conceive through IVF. MAIN OUTCOME MEASURE(S) Pregnancy rates (PRs) and pregnancy loss rates relative to each of the various antiphospholipid antibodies that were measured. RESULT(S) There were 528 pregnancies for an overall PR of 66%. Pregnancy rates were equal among patients with positive and negative antiphospholipid antibodies for each of the 21 measured antibodies. Use of receiver operator characteristic curves and logistic regression further confirmed that there was no relationship between PRs or outcome based on antiphospholipid antibodies for any definable threshold value. CONCLUSION(S) Elevated antiphospholipid antibody levels are not associated with any change in PRs or pregnancy loss rates in patients attempting to conceive through IVF.

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.

Rebuttal: interooplasmic transfers in humans

There have been two known instances of chromosomal abnormalities in clinical pregnancies resulting from ooplasmic transplantation in our work (Barritt et al., 2000). One singleton pregnancy ended in a spontaneous miscarriage in the first trimester. The fetus was karyotyped as 45,XO. The rate of spontaneous miscarriage in assisted reproduction is 12–15% and such miscarriages (as in the natural population) are by standard practice not included in the calculation of congenital abnormalities (Simpson, 1990). Another ooplasmic transplantation pregnancy was analysed by ultrasonography at 15 weeks and shown to be a twin gestation with one fetus developing abnormally. Amniocentesis and analysis revealed the presence of a 45,XO karyotype. This fetus was electively reduced and the second 46,XX fetus was subsequently delivered normally. The 45,XO karyotype is the most common chromosomal abnormality associated with abnormal developmental morphology at the time of ultrasonography (Byrne et al., 1985). As much as 70% of spontaneous first trimester miscarriages are associated with chromosomal abnormalities and 45,XO is the most common single chromosome abnormality within this group with an incidence of 20–25% (Strom et al., 1992; Simpson, 1990) The overall incidence of a 45,XO karyotype in amniocentesis and chorionic villus sampling analysis is approximately 1/500 and 1/250 respectively (Gravholt et al., 1992). Press reports suggesting that the 45,XO karyotype is “rare” or “mysterious” are obviously distortions of the facts.

Mitochondrial DNA deletions in primate embryonic and adult stem cells

Mitochondrial DNA (mtDNA) mutations occur naturally in skeletal muscle fibers from aged rhesus macaques. In addition, mtDNA mutations have been observed in germinal vesicle oocytes from fertile monkeys. The goal of this study was to determine whether the rhesus macaque mitochondrial common deletion was present in oocytes and embryos generated by in-vitro embryo production (IVP), as well as in rhesus adult and embryonic stem cell lines. The rhesus common deletion was detected in IVP-generated embryos, three IVP-derived embryonic stem cell lines (ORMES 1, 2 and 7), one in-vivo-derived embryonic stem cell line (R4) and multiple passages of an adult bone marrow stromal cell (BMSC) line. Mitochondrial DNA from an adult adipose stromal cell (ATSC) line was compared with mtDNA from an immortalized line transfected with a retroviral vector expressing telomerase, ATSC-TERT. Multiple passages of the ATSC line harboured a dramatically higher level of the rhesus common deletion than the immortalized ATSC-TERT line. Accumulation of mtDNA mutations in oocytes, embryos and subsequent embryonic stem cell lines, as well as adult stem cell lines, may contribute to mitochondrial dysfunction, and thereby impair ATP production. The authors believe this information establishes a compelling argument for the parallel development of embryonic stem cell technology in non-human primates and humans.

Genotyping: the HLA system and embryo development*

The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of maternal pre-eclampsia. HLA-G has 15 known alleles at the DNA level, and allelic frequency varies among ethnic groups. This study describes the results of an inaugural attempt to correlate an HLA-G genetic polymorphism with pregnancy outcome in a patient population undergoing IVF. The study group was composed of 102 Caucasian women. A maternal HLA-G genetic polymorphism was investigated by polymerase chain reaction (PCR) analysis of DNA collected from granulosa cells surrounding oocytes harvested for the IVF procedure. While no statistically significant correlation was identified in this initial study, larger studies examining DNA from trios of mother, father and offspring are planned.

128 MITOCHONDRIAL DNA DELETIONS IN RHESUS MACAQUE OOCYTES, EMBRYOS, AND ADULT AND EMBRYONIC STEM CELLS

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was >3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates. This study was supported by National Institutes of Health grants RR15395 and HD045966.