It may also be possible to increase oocyte energy after ovulation. Cohen and colleagues in the late 1990’s demonstrated improved embryo development and live births with ooplasm injection (16). This work was based on the observation of the 2-cell embryo block in some mice that could be overcome by injection of cytoplasm from the oocytes of a strain of mice without a 2-cell block (17). In patients with poor embryo development and previous failed IVF cycles, injection of a small volume of ooplasm from young healthy egg donors resulting in improved embryo development and implantation leading to ongoing pregnancy. The children born following this procedure were healthy but many were found to have mitochondrial DNA heteroplasmy (18,19) indicating that the donor ooplasm contained mitochondria and suggesting that the donor mitochondria may have been responsible for the increased energy and improved embryo development (20). Because mtDNA heteroplasmy is considered to be altering the fetal genome, potentially for future generations, ooplasm transfer from donor oocytes has not been sanctioned by the US and Canada.

 

In an attempt to increase embryo development without altering the genome of the embryo, Jurisicova and colleagues studied the potential utility of microinjection of recombinant mitochondrial proteins. In a murine model of embryo arrest, we showed that HTF, a suboptimal culture medium, induced 2-cell arrest in a subset of embryos and compromised preimplantation embryo development in others when compared to KSOM medium (21). Rates of blastocyst formation at about 96 hours in HTF culture and total cell number in blastocysts were dramatically reduced, while the cell death index (CDI) was elevated compared to embryos in KSOM. The poor quality of embryos was also reflected by nuclear staining (DAPI), showing smaller blastocysts with multiple apoptotic cells. Finally, expression of BCL-X protein was decreased in 2-cell embryos cultured for 24 hours in HTF medium (21). Using this model, we microinjected the modified cell death protector protein, recBCL-XL missing the transmembrane domain (ΔTM) into zygotes using a robotic system and the effect on early embryo development in HTF medium was determined (22). The ability of mouse zygotes to progress through development and form blastocysts in suboptimal HTF medium was significantly increased upon microinjection of recBCL-XL (ΔTM) protein (n=424) when compared to buffer injected embryos (n=414). In addition, total cell number per embryo was significantly increased and cell death index (CDI) was decreased as determined by nuclear counterstaining (DAPI) images of blastocysts at day 4.5. Injection of recBCL-XL (ΔTM) also significantly reduced the accumulation of ROS in the mouse embryos (22). This procedure prevents any alteration of the genome since the injected proteins will be diluted out and become non-detectable after several cell divisions. It remains to be determined if injection of mitochondrial proteins associated with anti-apoptosis will be of benefit to human embryos in women with a history of poor embryo development and failed IVF cycles.

More recently, we have modified the donor ooplasm injection approach in an attempt to improve embryo development by microinjecting autologous human mitochondria derived from the patient’s own oocyte precursor cells into human oocytes at the time of ICSI. In 2004, Jonathan Tilly and his lab discovered egg precursor cells (EggPCs) found in the avascular epithelium of the ovarian cortex (23,24). These EggPCs could be isolated after taking a superficial laparoscopic biopsy of the ovarian cortex using scissors. The tissue biopsy was frozen and later thawed, digested enzymatically and the EggPCs identified and counted by fluorescence activated cell sorting performed using antibodies specific to human VASA, a cell antigen found both in and on EggPCs (24). These EggPCs were found to contain mitochondria that were morphologically similar to mature oocyte mitochondria (Figure 1). We believed that these EggPC mitochondria could provide an autologous source of mitochondria for injection into energy deficient oocytes, thereby bypassing the concerns that led to sanctioning of donor oocyte mitochondrial injection.