Martin Wilding

Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa

PurposeWe 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.MethodsIndividual 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.Results64.8% 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.ConclusionsComputerised selection of spermatozoa during ICSI procedures deselects physiological abnormal spermatozoa and improves clinical results.

Chaotic mosaicism in human preimplantation embryos is correlated with a low mitochondrial membrane potential.

OBJECTIVE To determine the relationship between the intrinsic mitochondrial deltapsi of human embryos and the embryo karyotype. DESIGN Analysis of mitochondrial deltapsi of living embryos followed by chromosomal enumeration with fluorescence in situ hybridization. A tertiary center for assisted reproduction technology. PATIENT(S) Fifty-two patients attending the fertility center for assisted reproduction. INTERVENTION(S) Donated embryos were loaded with a mitochondrial deltapsi-sensitive fluorescence dye. MAIN OUTCOME MEASURE(S) Mitochondrial deltapsi was measured by confocal microscopy. Subsequently, embryos were fixed and fluorescence in situ hybridization analysis was used to denote embryo karyotype. MAIN OUTCOME MEASURE(S) Mitochondrial deltapsi and embryo karyotype. RESULT(S) An association was observed between low mitochondrial deltapsi and the detection of chaotic mosaicism. Analysis of oocytes suggested that this was due to the effect of low mitochondrial deltapsi on the morphology of the meiotic apparatus. CONCLUSION(S) The data suggest that the intrinsic mitochondrial deltapsi of human oocytes programs the developmental fate of embryos through an effect on the ability of oocytes to form a normal meiotic apparatus and not through nondisjunction.

Imaging the spatial dynamics of calmodulin activation during mitosis

BACKGROUND Calcium is an important and ubiquitous signalling ion. In most cell types, changes in intracellular calcium concentrations are sensed by calmodulin, a signal transduction protein that regulates cell function through its interactions with kinases and phosphatases. Calcium signals show complex spatiotemporal patterning, but little, if anything, is known about the patterns of calmodulin activation inside cells. RESULTS We have measured calmodulin activation continuously during mitosis in living cells with a new probe, a fluorescent adduct of calmodulin termed TA-calmodulin. We found that calmodulin was activated locally and episodically in the nucleus and mitotic spindle. The pattern of calmodulin activation was different from the pattern of calcium signals and could not be predicted from the pattern of calcium increase. Calmodulin activation was essential for mitotic progression: both entry into mitosis and exit from mitosis were blocked by a novel peptide that bound to calmodulin with high affinity and so prevented the interaction of calmodulin with its target proteins. CONCLUSIONS These data suggest that calmodulin regulates mitotic transitions and demonstrate the utility of fluorescent adducts for studying protein activation in living cells with good temporal and spatial resolution.

Effects of recombinant LH (rLH) supplementation during controlled ovarian hyperstimulation (COH) in normogonadotrophic women with an initial inadequate response to recombinant FSH (rFSH) after pituitary downregulation

background  This study was aimed to evaluate the effect of different recombinant LH (rLH) doses on the ovarian outcome of normogonadotrophic women with an initial inadequate response to recombinant FSH (rFSH) after pituitary downregulation.

Mitochondria and human preimplantation embryo development

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.

An oocyte score for use in assisted reproduction.

PurposeIn 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.MethodsThe 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%.ConclusionsClinical 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.

Energy substrates, mitochondrial membrane potential and human preimplantation embryo division

Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate. Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human. This is mainly due to the lack of human embryos for research and the reliance on animal model systems. In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division. The mitochondrial membrane potential was measured with fluorescence techniques. Cell division was scored as the number of blastomeres on day 3. Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources. The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media. Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential. However, cell division continued in the presence of glucose, suggesting that some energy production can continue. These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development. The role of lactate is as yet unclear.

How do spermatozoa activate oocytes

Although the spermatozoon is 500,000 times smaller in volume than the oocyte, it induces rapid and dramatic changes in oocyte physiology that lead to meiosis re-initiation. These oocyte activation events are described here, as is the evidence for a soluble activating factor in the spermatozoon. Since changes in plasma membrane conductance, calcium ion release and maturation-promoting factor inactivation are common to all animal oocytes at activation, it is expected that the sperm-borne trigger is also ubiquitous. One likely candidate, phospholipase C (PLC) zeta 1, induces calcium release in mammalian oocytes; however, work on other deuterostomes suggests that the sperm factor is non-specific and multifactorial, regulating several activation events. Human, sea urchin and ascidian gametes are remarkably similar and comparative studies across the deuterostomes may help in elucidating basic principles in fertilization. Questions to be answered include the identification of PLC zeta 1 in invertebrate spermatozoa and the characterization of other targets in mammalian oocytes, such as the adenosine diphosphate ribose/nitric oxide pathway.

Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction

ObjectivesTo compare embryo survival, pregnancy and implantation rates after cryopreservation of human cleavage-stage embryos with slow-rate cryopreservation or vitrification.Study design262 patients, attending for assisted reproduction, were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols. Excess embryos were cryopreserved on day 3 either by vitrification, or slow-rate cryopreservation in a programmable freezer. Cycles of thawing were monitored for thaw efficiency, pregnancy and implantation rates.ResultsClinical pregnancy and implantation rates were highly comparable between cycles in which day 3 embryos were thawed either after slow-rate cryopreservation or vitrification.ConclusionsThese data suggest that vitrification of human embryos during assisted reproduction cycles achieves comparable success rates to fresh cycles and therefore can be applied in the laboratory of assisted reproduction.

The Effect of Ease of Transfer and Type of Catheter Used on Pregnancy and Implantation Rates in an IVF Program

AbstractPurpose: To test the effects of type of embryo transfer catheter, transfer difficulty, and observations after the transfer procedure on pregnancy and implantation rates in an IVF programme. Methods: Patients were prepared for IVF using standard protocols. Embryo transfer was performed using either Edwards-Wallace or TDT catheter. The difficulty of transfer was graded by a clinician and biologist. Blood observed inside the catheter after the transfer procedure was scored as endometrial damage. Pregnancy and implantation rates were scored. Results: Type of embryo transfer catheter and the observation of blood did not significantly affect pregnancy and implantation rates when transfer was performed by a single operator. Conclusions: In the hands of experienced, skilled operators, neither choice of transfer catheter and difficulty of transfer nor observations of blood on the transfer catheter caused any significant reduction in outcome to the patient.