Henry Malter

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.

Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology

OBJECTIVE To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. DESIGN Two studies were performed: partial zona dissection and IVF was applied in 57 couples during the first period, and subzonal sperm insertion was also applied in a second group of 47 couples. SETTING Procedures were performed in an academic research environment. PATIENTS, PARTICIPANTS Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. INTERVENTIONS Oocytes were micromanipulated with either partial zona dissection or subzonal sperm insertion, or the zona pellucida was left intact. Embryos were replaced in patients prophylactically treated with methylprednisolone and antibiotics. MAIN OUTCOME MEASURES Because several microsurgical fertilization techniques are now available, this study was performed to compare sperm parameters, embryo morphology, fertilization, and implantation rates after application of two successful micromanipulation procedures. RESULTS Twenty-one pregnancies were established in 104 patients, 5 definitely from subzonal sperm insertion and 4 from partial zona dissection. Patients who failed IVF before had a similar chance of pregnancy after the use of micromanipulation, as first time patients (9/53 versus 12/51). In a subgroup of 15 patients who failed IVF with insufficient numbers of motile sperm, fertilization was significantly higher after subzonal sperm insertion. Partially zona-dissected embryos from couples with severe teratozoospermia (less than or equal to 5% normal forms; strict criteria) had significantly more morphological abnormalities than those from patients with moderate teratozoospermia (6% to 10% normal forms). In severely teratozoospermic patients, significantly fewer partially zona-dissected than subzonally inserted embryos implanted. CONCLUSIONS The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.

Expression profiles of individual human oocytes using microarray technology.

Microarray technology is a relatively new technique that provides the investigator with the ability to monitor and quantify the expression of thousands of genes simultaneously. This technological breakthrough has the potential to provide detailed insight into cellular processes involved in the regulation of gene expression. In this study, microarray methods were used to examine the expression of linearly amplified RNA from individual and pooled (n = 5) human oocytes. The amplification strategy consistently produced a complex representative cDNA population. A catalogue of 1361 transcripts expressed in human oocytes was identified, of which 406 have been independently confirmed using other methods.

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.

Fixing oocytes? A bovine model provides new hope.

In a previous issue of Reproductive BioMedicine Online, Chiaratti and co-workers presented a bovine model for ooplasmic transfer, which demonstrated a positive effect on early development. Developmental deficits resulting from artificial treatment of recipient eggs with a toxic compound were ameliorated by the addition of small volumes of healthy donor cytoplasm. This model provides an important advance in the understanding of ooplasmic effects in early development and addresses issues about the prior human trials in this area.

Combinatorial peptide library binding of mammalian spermatozoa identifies a ligand (HIPRT) in the axin protein: putative identification of a sperm surface axin binding protein and intriguing developmental implications

The identification of components in cell-cell interactions is an important research goal in reproductive and developmental biology. Such interactions are critical to gamete development, fertilization, implantation and basic development. Several proteins involved with sperm-oocyte interaction and other developmentally important phenomena have been identified. However, these are obviously only a subset of the molecular components involved in such complex cell-cell interactions. One method that has been used to identify binding partners for particular molecular targets is the use of combinatorial libraries accessible on phage surfaces. For the most part, this technique has mainly been applied to screen specific target moieties. However, in some cases whole-cell screening has been attempted. This study describes the first report of screening intact, living mammalian gametes using a proprietary whole-cell combinatorial library binding and analysis protocol. Results from the first screening protocol of mouse spermatozoa strongly identified a putative sperm-binding ligand using proprietary bioinformatic analysis. This amino acid sequence (HIPRT) precisely corresponds with a previously characterized highly conserved protein-protein interaction site in the axin protein. This sequence is found within the binding site for a known sperm surface protein, glycogen synthase kinase-3. This result not only provides proof of the utility of this technique to identify cell surface ligands in mammalian gametes, but it also suggests a potential role for spermatozoa in facilitating developmental axis formation in mammalian embryos.

The first clinical nuclear transplantation in China: new information about a case reported to ASRM in 2003

Before there was public debate on mitochondrial replacement therapy (MRT) to treat mothers at risk of transmitting mitochondrial disease (Hyslop et al., 2016), and before there was a publicly traded company called OvaScience aiming to use nuclear transplantation and stem-cell technologies to treat infertility (Woods and Tilly, 2015), there was a solitary case report from China about the use of nuclear transplantation with donor oocyte-derived cytoplasm at the zygote stage to overcome cleavage arrest in a patient’s embryos (Zhang et al., 2003). The landmark abstract, presented to the 2003 annual meeting of the American Society for Reproductive Medicine (ASRM), was received with excitement and concern, sentiments that promptly seeped into the lay press. This was not surprising considering the topic and the risk-sensitive audience of reproductive scientists listening to the presentation given by the lead author, Dr John Zhang. The case report described an IVF patient with normal fertilization whose zygotes divided only once. Early development arrest affected all the embryos of this patient in two consecutive cycles. Approximately 10% of cleaving embryos arrest in culture, but the repeated arrest of an entire cohort is indeed rare, and, based on experimental evidence, could be attributable to ooplasmic deficits. Ooplasmic developmental determinants are far from understood, but one possibility is the deregulation of proteins like the stress sensor p66Shc, which may affect signaling pathways for mitochondrial function (Betts and Madan, 2008). Complete developmental arrest represents an extrememanifestation of ooplasmassociated deficits that can occur during human assisted reproduction treatment cycles. The idea that ooplasmic deficits could be overcome by cytoplasmic replacement or augmentation using donor oocytes or embryos is based on work performed nearly three decades ago by Pratt and MuggletonHarris (1988) in the mouse. These authors injected 8 pl of cytoplasm extracted from normally dividing 2-cell mouse embryos into single blastomeres of arrested 2-cell recipient embryos. Using various combinations of blocking and nonblocking mouse strains, 2-cell embryos were injected with the aid of a membrane relaxant in order to avoid lysis. The authors’ aim was to study cell-cycle dependent blockage. They found that only 4% of the total donor blastomere cytoplasmic volume was sufficient to overcome the block. The ability of a small volume of presumably normal cytoplasm to change cell cycle events in early embryos became the basis for cytoplasmic transfer to treat oocytes of patients with multiple cycles of abnormal embryo development, using ooplasm from donor eggs during ICSI (Cohen et al., 1998). A similar approach has been recently advocated using autologous transfer of mitochondria derived from egg precursor cells (AUGMENTTM, OvaScience), which morphologically appear to resemble ooplasmic mitochondria (Woods and Tilly, 2015). In a follow-up paper by Zhang and coworkers in this issue of RBMOnline (Zhang et al., 2016) the case performed in China in 2003 is presented in more detail. Why the 13-year hiatus between presentation of the abstract at a meeting and publication of a full paper? Perhaps the authors were discouraged by the tragic loss of the resulting triplet pregnancy and the criticism from colleagues and ethicists. Whatever the circumstances, the current paper describes the reconstitution protocol and the DNA analyses which showed that the fetuses had mitochondrial DNA from the oocyte donor and nuclear DNA from the mother. Unfortunately, other details are missing: no micrographs are provided of the nuclear transplantation procedure and there is essentially no data or micrographs from the two unsuccessful treatment attempts that led the authors to apply nuclear transplantation in the first place. None of the patient’s zygotes were kept in culture as a control in parallel with the reconstituted zygotes in order to document repeated early arrest. Other questions remain unanswered about this case. What was the review and ethics approval process at Sun-Yat Sen University where this experiment was conducted? Was the demise of one fetus following premature rupture of membranes at 24 weeks of gestation, and cord prolapse and demise

Enhancement of Fertilization and Hatching Using Micromanipulation

Over 50 babies have now been born following the application of micromanipulation to oocytes and embryos. The current proceedings deal almost uniquely with the genetic diagnosis of biopsied polar bodies, blastomeres and nuclei from blastomeres or trophoblastic cells, however, micromanipulation procedures in assisted reproductive technology are performed for a variety of other reasons. Although the purpose of preimplantation diagnosis may differ from that of other microsurgical technologies, these methods have in common that the zona pellucida is breached. When normal fertilization is absent due to abnormalities in the sperm or oocytes, micromanipulation has been used to promote sperm-egg fusion (Malter and Cohen, 1989a; Ng et al, 1990). When Polyspermic fertilization occurs, micromanipulation may be used to return the zygote to a genetically normal, viable state (Gordon et al, 1989; Malter and Cohen, 1989b). Also, micromanipulation has been used to promote implantation by assisting the hatching process (Cohen et al, 1990a). However, micromanipulation imposes artificial conditions on the gametes and embryos.

Techniques for Microsurgical Fertilization

During the past two years, pregnancies have been reported following the use of two microsurgical fertilization techniques: Partial Zona Dissection (PZD) and Subzonal Insertion (SI or MIST for microinsemination sperm transfer) (Cohen et al, 1988; Ng et al, 1988). To date, 25 pregnancies have been established with 12 healthy babies born from the application of PZD in 7 clinics worldwide. The pregnancy and birth rates from SI are not definite, although at least four pregnancies and one birth have been reported (Ng et al, 1990; Fishel et al, 1990).