Most human embryos cease developing before implantation, due to a variety of deficiencies in genetic constitution, metabolism or cytoplasmic defects. The success of infertility treatment by in vitro fertilisation (IVF) depends upon selecting embryos that do have the potential to develop and using them for patient treatment.
We are exploring ideas to improve basic understanding of the key features of a competent oocyte or embryo, using a range of methods from detailed molecular analyses to quantitative imaging of live cells.
Analysis of telomere length
Telomeres are repeated DNA sequences that protect the ends of chromosomes. They are important for normal meiosis and cell division. Moreover, they control cell ageing and longevity by shortening slightly with each cell division. At a certain critically short length, cellular senescence occurs. Cells that will undergo many divisions therefore require longer telomeres to start with, or a means of maintaining telomeres, such as the enzyme telomerase. Telomere extension mechanisms are restricted to stem cells and those that have transformed to become 'immortal'.
We are studying telomere length in human embryos, sperm, oocytes and embryonic stem cells in order to understand whether their length can be used to test the quality of cells intended for clinical use.
- Wong H P, Yding Andersen C, Hartshorne G M (2008) Telomere length of human embryonic stem cells measured by Flow–FISH, UK Stem Cell Network Inaugural Meeting, 9-11 April 2008, Edinburgh, Abstract
- Turner S, Wong HP, Rai J, Hartshorne GM. (2010) Telomere lengths in human oocytes, cleavage stage embryos and blastocysts. Mol Hum Reprod. 2010 Sep;16(9):685-94. Free text
Telomeres in a human embryo interphase nucleus. Telomeres are highlighted with a quantitative fluorescent PNA probe (green), the intensity of which is proportional to telomere length. Chromatin is stained blue with DAPI.
Quantitative image analysis
Any viability test used clinically should ideally be non-invasive. We undertake quantitative image analysis of living cells, for example measuring oocyte diameter, spindle location or orientation of pronuclei, and relate the results to the observed potential of the oocytes/embryos in vitro or in assisted conception treatments.
- Woodward B, Montgomery S, Hartshorne G M, Campbell K, Kennedy R (2008) Spindle position assessment prior to ICSI does not benefit fertilization or early embryo quality, Reprod Biomed Online, Feb 2008: 16 (2); pp 232-8
- Cavilla J L, Kennedy C R, Byskov A G, Hartshorne G M (2008) Human immature oocytes grow during culture for IVM, Human Reproduction, Jan 2008: 23 (1); pp 37-45 Free text
- Garello C, Baker H, Rai J, Montgomery S, Wilson P, Kennedy C R, Hartshorne G M (1999) Pronuclear orientation, polar body placement & embryo quality after ICSI and IVF - further evidence for polarity in human oocytes? Human Reproduction: 14; pp 2588-2595 Free Text
Measuring the orientation of pronuclei in relation to the polar bodies in human pronuclear oocytes. Figure from Garello et al, 1999.
Identification and modelling of molecular biomarkers
We use fluorescent markers to highlight DNA, proteins or other key components of the embryo or oocyte. Modifying the conditions under which the oocyte or embryo is cultured may affect its development and also expression of the marker of interest, providing insights into developmental control. For example, markers of oxidative stress can provide evidence of an embryo’s metabolic state. Markers of DNA methylation may provide insights into the control of methylation as a means of gene silencing in early embryos. Similar methods have been applied to study isolated ovarian follicles in vitro.
We are currently moving towards computer modelling and virtual reality systems to expand our current analyses to three dimensions.
- Beaujean N, Hartshorne G, Cavilla J, Taylor J, Gardner J, Wilmut I, Meehan R, Young L (2004) Non-conservation of mammalian preimplantation methylation dynamics, Current Biology, Apr 6, 14 (7), R266-R267
- Mitchell L M, Kennedy C R, Hartshorne G M (2004) Pharmacological manipulation of nitric oxide levels in mouse follicle cultures demonstrates key role of extrafollicular control of ovulation, Human Reproduction, 19 (8); pp 1705-1712 Free Text
Section through a human blastocyst (6 days) using confocal microscopy. Stained for mitochondrial redox potential (red/green) and chromatin (blue). The inner cell mass (ICM) is the collection of cells on the right which form the embryo proper. The ICM is surrounded by flattened polar trophectoderm cells on the right and mural trophectoderm on the left. The trophectoderm eventually produces the extra-embryonic membranes. Notice the abnormal cell at the bottom of the inner cell mass, having multiple condensed nuclei.
Group leader and key contact
Prof Geraldine Hartshorne Professorial Fellow