Mitochondria play a vital role in the metabolism of energy-containing compounds in the oocyte cytoplasm to provide adenosine trisphosphate for fertilization and preimplantation embryo development. In this study, ratiometric confocal microscopy with the mitochondrion-specific membrane potential-sensitive fluorescence dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) was used to measure the activity of mitochondria in human oocytes and developing preimplantation embryos. Mitochondria in oocytes and embryos were characterized by distinct localized aggregation patterns. These patterns however did not determine localized regions of heterogeneity in mitochondrial activity. Mitochondrial activity was analysed during oocyte maturation and after fertilization. The activity of mitochondria in fresh metaphase II oocytes was negatively correlated with maternal age. This trend continued when the activity of developing embryos was analysed. Mitochondrial activity was strongly correlated with the rate of embryo development on day 3 after fertilization, but not on day 2. Partial regression analysis showed that the rate of cleavage of preimplantation embryos was more highly correlated with embryo mitochondrial activity than maternal age. These data suggest that the efficiency of mitochondrial respiration in oocytes and preimplantation embryos is closely correlated with the programmed rate of embryo development, and suggest that maternal age further influences this factor. The loss of mitochondrial activity in oocytes obtained from ageing couples may therefore contribute to lower embryo development and pregnancy rates observed during cycles of IVF.

Purpose We used computer assisted sperm selection (MSOME) during cycles of intracytoplasmic sperm injection to test whether this technique improves results over traditional ICSI protocols. We also used the TUNEL assay to test whether MSOME could deselect physiologically abnormal spermatozoa. Methods Individual spermatozoa were examined with MSOME. Normal and abnormal spermatozoa were tested for the level of DNA fragmentation using TUNEL assay. In a prospective, randomized trial, patients were selected for standard ICSI, or IMSI techniques. We tested the two groups for biological and clinical parameters. Results 64.8% of spermatozoa, otherwise selectable for ICSI, were characterized by abnormalities after computer-assisted sperm analysis. These sperm were also characterized by an increase in the level of DNA fragmentation. We noted an increase in embryo quality, pregnancy and implantation rates after computerized sperm selection during ICSI procedures. Conclusions Computerised selection of spermatozoa during ICSI procedures deselects physiological abnormal spermatozoa and improves clinical results.

We have used fluorescence techniques to study the regulation of intracellular pH during fertilization and preimplantation embryo development in human oocytes. The intracellular pH of human oocytes during maturation and fertilization was always 7.4, suggesting that these processes do not involve long-term changes in intracellular pH. The recovery of intracellular pH of human oocytes and embryos after extracellular acid or alkaline shock was investigated. Fresh metaphase II oocytes and preimplantation embryos showed similar rates of recovery following alkaline shock. However, aged and immature oocytes had a significantly slower rate of recovery to physiological pH. Early embryos up to the morula stage were unable to recover following acidosis, whereas blastocysts could control both acidosis and alkalosis. We assessed the sensitivity of fertilization and early development in the human to extracellular pH. Our results show that insemination in the human is pH-sensitive, whereas intracytoplasmic injection (ICSI) activated oocytes at all pH tested. We suggest that this is due to the pH-sensitivity of the sperm-zona pellucida interaction, which is bypassed during the ICSI procedure. Further development in human embryos is more sensitive to alkalinity than acidity. We discuss these results in terms of the extracellular pH in vivo in the female reproductive tract.

No abstract

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

Purpose In this work, we describe a system for the morphological scoring of human oocytes prior to fertilisation and use this system to test whether oocyte morphology is an indicator of fertilisation, embryo development and implantation potential. Methods The study is a prospective trial of the use of oocyte morphological scores in 822 patients undergoing their first cycle of ICSI. Analyses of oocytes were performed prior to ICSI procedures and the scores compared with fertilisation rates, embryo quality and clinical results.Results 'Top quality' oocytes had a significantly higher level of fertilisation (96%) as compared to low scoring oocytes (25.6%). Where top quality oocytes formed top quality embryos, we noted a clinical success rate of 63.4%. Conclusions Clinical success rates were increased in cases where top quality oocytes formed top quality embryos after ICSI. The analysis of oocyte morphology may represent a positive selection feature during ICSI.

Couples suffering from idiopathic infertility are among the most difficult to treat in IVF cycles due to the unknown cause of failure to reproduce. This can lead to years of unsuccessful attempts to conceive and often the abandonment of treatment. In some couples, the only observable problem is the development of poor quality embryos. In this case report, we describe the successful use of cytoplasmic donation in a couple where the high level of embryo fragmentation and poor embryo development was thought to be the unique cause of failure to conceive after intracytoplasmic sperm injection (ICSI). We noted a strong reduction in embryo fragmentation and a consequent increase in embryo quality after the treatment. Transfer of four of the embryos receiving donor cytoplasm led to a pregnancy with two gestational sacs and the birth of healthy twins.

The 'maternal age effect' in human reproduction, characterized by a negative relationship between maternal age and reproductive efficiency, remains a poorly understood phenomenon. Current data suggest that oocyte physiology determines this relationship. In this review, we present a hypothesis of a mitochondrial role in the physiology of ageing in human oocytes. We suggest that the efficiency of oxidative phosphorylation in the ageing human oocyte is degraded by free radical attack on the primordial oocytes residing in the ovary. Although deficiencies in oxidative phosphorylation can be accounted for in the short term by anaerobic respiration, we suggest that, in the long term, the level of oxidative phosphorylation strongly influences oocyte quality.