2) Electrophysiology to check embryo viability.
   In the field of electrophysiology, it is well known that the resting potential (RP) of a cell may be used to give a clear idea of cell viability. For instance the RP of a healthy early mouse embryo is about -25 to -40 mV (Lee, 1987), whereas a damaged (“leaky”) embryo will only record about -10 to -20 mV (Powers & Tupper, 1974; Hagiwara & Jaffe, 1979). By using simple electrophysiological recording equipment (Lee, 1987) it is possible to make recordings from embryos without damaging them and to subsequently transfer them in utero and to obtain live birth. The electrodes used for recording are finer than a human hair (Purves, 1981; Thomas, 1978) and have an even smaller diameter to ICSI microinjection pipettes which are routinely used for injecting sperm into ova without detriment.

Aims of project
1) To routinely record from human embryos with a view to documenting RPs  and to determine whether their is clear correlation between negatively high potentials and embryo viability. Recording equipment and microelectrodes will be mounted on the ICSI equipment. Recordings will be made just prior to embryo transfer.

3) Blastocyst culture to assess viability
 In many countries outsideof the UK, blastocyst culture and transfer has become a popular variation of IVF. New culture media purportedly advantageous for the culture of blastocysts (5 day old embryos) have been developed. In the USA, reports suggest that up to 40% success rates are possible by units, which perfect this method.  It is believed that adoption of this method puts up to 15% of all couples at risk of failing to reach embryo transfer. Prior to starting such a programme, trying 3 day and 4 day culture is highly recommended beforehand. Frozen embryos will be used to trial days 3 to 5 culture in order to perfect the method beforetrialling it with embryos destined for transfer into paatients.

 
4) Implantation study with TC Li in Sheffield.

 Implantation remains a topic of great mystery. Assisted hatching deals with one of the obstacles of implantation, but why an embryo which is able to hatch and which is potentially viable, still fails to implant remains a mystery. The possibility that embryos signal the endometrium, bringing about profound changes in it, needs to be studied. TC Li in Sheffield has published a great deal on the endometrium. In 1993-1994 TC and Sammy Lee held a rare research licence from the HFEA to carry out human embryo implantation studies on human endometrium grown in culture. Donated embryos may be used to continue this research.
5) Immunological study with  the Rheumatology department at UCH Middlesex Hospitals.
 Work done in collaboration with the Rheumatology department demonstrates that fertility treatment may affect the antibody producing cells in fertility patients. Donated embryos may be used to study how  antibodies may affect human embryos and their development.
6) Study of gap junctionsin human embryos.
   Pregnancy rates following in-vitro fertilization (IVF) and subsequent embryo transfer are low, averaging only I7% per cycle in the UK in 1998 (Human Fertilisation and Embryology Authority, 1999). It has been suggested that a large proportion of embryonic loss occurs during preimplantation stages (Hardy, 1993). While 95% of human zygotes successfully undergo the first two cleavage divisions following IVF, ~60% of preimplantation embryonic arrest in vitro after the 8-cell stage, after compaction and up to blastocyst formation (Hardy et al., 1996).
  In the preimplantation mouse embryo, it has been demonstrated that good gap junctional communication is essential for the maintenance of compaction and subsequent preimplantation development (Buehr et al., 1987; Lee et al., 1987; Becker et al., 1995). In the early embryo cells are linked by gap junctions, intercellular structures that allow the transfer of ions and small molecules directly from one cell to the next (review by Becker & Davies, 1995). The cells of the early mouse embryo are linked by morphologically recognizable gap junctions from the 8-cell stage onwards, when the embryo begins to compact (Lo & Gilula, 1979; Lee et al, 1987). The proteins that make up these gap junctions can be recognized by immunocytochemical staining of the constituent gap junction (connexin) proteins from the 8-cell stage onwards. These issues may be addressed by raising antibodies to peptides derived from connexin43, since connexin43 is important as the predominant connexin protein expressed at the 8-cell stage in the mouse embryo and it is known that communication through gap junctions containing connexin43 is essential for the maintenance of compaction (Becker et al., 1995; Hardy et al., 1996).

Aims of project
1) Embryo biopsy and staining of blastomeres with antibodies raised to connexin43. These studies will help to determine optimum distribution of gap junctions in blastomeres derived from 8 cell human embryos, thereby enabling us to predict embryo viability.