J. Barritt

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.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis

BACKGROUND This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

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.

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.

Paternal age and assisted reproductive technology outcome in ovum recipients

This study suggests that paternal age may be inversely associated with reproductive outcome, as demonstrated by a decline in fertilization, blastocyst formation, implantation and cryopreservation rates with advancing age.

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.

Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization

A major challenge of assisted reproduction technologies (ARTs) is to mimic the natural environment required to sustain oocyte and embryo survival. Herein, we show that the ceramide‐metabolizing enzyme, acid ceramidase (AC), is expressed in human cumulus cells and follicular fluid, essential components of this environment, and that the levels of this enzyme are positively correlated with the quality of human embryos formed in vitro. These observations led us to develop a new approach for oocyte and embryo culture that markedly improved the outcome of in vitro fertilization (IVF). The addition of recombinant AC (rAC) to human and mouse oocyte culture medium maintained their healthy morphology in vitro. Following fertilization, the number of mouse embryos formed in the presence of rAC also was improved (from ~40 to 88%), leading to ~5‐fold more healthy births. To confirm these observations, immature bovine oocytes were matured in vitro and subjected to IVF in the presence of rAC. Significantly more high‐grade blastocysts were formed, and the number of morphologically intact, hatched embryos was increased from ~24 to 70%. Overall, these data identify AC as an important component of the in vivo oocyte and embryo environment, and provide a novel technology for enhancing the outcome of assisted fertilization. Eliyahu, E., Shtraizent, N., Martinuzzi, K., Barritt, J., He, X., Wei, H., Chaubal, S., Copperman, A. B., Schuchman, E. H. Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization. FASEB J. 24, 1229–1238 (2010). www.fasebj.org

Nitrogen vapor shipment of vitrified oocytes: time for caution

Oocyte cryopreservation for fertility preservation, in both medical and elective situations, has significantly increased as freezing technology has improved. Slow freezing techniques demonstrated ∼ 50-80% survival of mature oocytes, however vitrification with ∼ 97% survival has become the preferred method for oocyte cryopreservation around the world. Our work investigated the effect of transporting cryopreserved oocytes to and from a long-term storage facility. Our findings demonstrate that extra caution should be practiced for vitrified oocytes, especially when handling and transferring between shipping and long-term cryopreservation storage containers.

Commercially available enhanced in vitro maturation medium does not improve maturation of germinal vesicle and metaphase I oocytes in standard in vitro fertilization cases

Improvements in in vitro maturation techniques have focused on culture optimization to increase oocyte maturation rates. Specialized culture media, now commercially available, did not perform significantly better than standard IVF culture media for maturation of immature oocytes in our normal IVF cases.

The morphology of extracted testicular sperm correlates with fertilization but not pregnancy rates

To investigate sperm morphology on the day of fresh testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI), and its effect on fertilization and pregnancy rates, as TESE in conjunction with ICSI results in high fertilization and pregnancy rates in most patients, but to our knowledge only one small study has assessed the morphology of retrieved sperm and found no correlation with the success of fertilization.