Oocyte and embryo evaluation by AI and multi-spectral auto-fluorescence imaging: Livestock embryology needs to catch-up to clinical practice.

A highly accurate 'non-invasive quantitative embryo assessment for pregnancy' (NQEAP) technique that determines embryo quality has been an elusive goal. If developed, NQEAP would transform the selection of embryos from both Multiple Ovulation and Embryo Transfer (MOET), and even more so, in vitro produced (IVP) embryos for livestock breeding. The area where this concept is already having impact is in the field of clinical embryology, where great strides have been taken in the application of morphokinetics and artificial intelligence (AI); while both are already in practice, rigorous and robust evidence of efficacy is still required. Even the translation of advances in the qualitative scoring of human IVF embryos have yet to be translated to the livestock IVP industry, which remains dependent on the MOET-standardised 3-point scoring system. Furthermore, there are new ways to interrogate the biochemistry of individual embryonic cells by using new, light-based methodologies, such as FLIM and hyperspectral microscopy. Combinations of these technologies, in particular combining new imaging systems with AI, will lead to very accurate NQEAP predictive tools, improving embryo selection and recipient pregnancy success.

Haemoglobin expression in in vivo murine preimplantation embryos suggests a role in oxygen-regulated gene expression.

Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage; in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.

Artificial blastocyst collapse prior to vitrification significantly improves Na+/K+-ATPase-dependent post-warming blastocoel re-expansion kinetics without inducing endoplasmic reticulum stress gene expression in the mouse.

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

A dual-mode mobile phone microscope using the onboard camera flash and ambient light.

Mobile phone microscopes are a natural platform for point-of-care imaging, but current solutions require an externally powered illumination source, thereby adding bulk and cost. We present a mobile phone microscope that uses the internal flash or sunlight as the illumination source, thereby reducing complexity whilst maintaining functionality and performance. The microscope is capable of both brightfield and darkfield imaging modes, enabling microscopic visualisation of samples ranging from plant to mammalian cells. We describe the microscope design principles, assembly process, and demonstrate its imaging capabilities through the visualisation of unlabelled cell nuclei to observing the motility of cattle sperm and zooplankton.

A novel embryo culture media supplement that improves pregnancy rates in mice.

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.

Maternal factors and the risk of birth defects after IVF and ICSI: a whole of population cohort study.

OBJECTIVE: To assess the contribution of maternal factors to major birth defects after in vitro fertilisation (IVF), intracytoplasmic sperm injection (ICSI), and natural conception. DESIGN: Retrospective cohort study in South Australia for the period January 1986 to December 2002. SETTING: A whole of population study. POPULATION: A census of all IVF and ICSI linked to registries for births, pregnancy terminations, and birth defects (diagnosed before a child's fifth birthday). METHODS: Odds ratios (ORs) for birth defects were calculated among IVF, ICSI, and natural conceptions for maternal age, parity, pre-pregnancy BMI, smoking, pre-existing diseases, and conditions in pregnancy, with adjustment for confounding factors. MAIN OUTCOME MEASURES: Birth defects classified by International Classification of Diseases (ninth revision) and British Paediatric Association (ICD9-BPA) codes. RESULTS: There were 2211 IVF, 1399 ICSI, and 301 060 naturally conceived births. The unadjusted prevalence of any birth defect was 7.1, 9.9, and 5.7% in the IVF, ICSI, and natural conception groups, respectively. As expected, the risk of birth defects increased with maternal age among the natural conceptions. In contrast, for IVF and ICSI combined, relative to natural conceptions, births to women aged ≤29 years had a higher risk (adjusted odds ratio, aOR 1.42; 95% confidence interval, 95% CI 1.04-1.94), births to women aged 35-39 years had no difference in risk (aOR 1.01; 95% CI 0.74-1.37), and births to women aged ≥40 years had a lower risk of defects (aOR 0.45; 95% CI 0.22-0.92). Defects were also elevated for nulliparity, anaemia, and urinary tract infection in births after ICSI, but not after IVF. CONCLUSIONS: The usual age-birth defect relationship is reversed in births after IVF and ICSI, and the associations for other maternal factors and defects vary between IVF and ICSI. TWEETABLE ABSTRACT: Risk of birth defects in women over 40 years is lower after infertility treatment than for natural conceptions.

Oocyte maturation and quality: role of cyclic nucleotides.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

STUDY QUESTION: Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? SUMMARY ANSWER: Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. WHAT IS KNOWN ALREADY: Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium.

STUDY QUESTION: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER: The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY: Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION: Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. LIMITATIONS, REASONS FOR CAUTION: Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence.

Recent studies have independently shown that cyclic adenosine 3'5'-monophosphate (cAMP) modulation prior to in vitro maturation (IVM) and epidermal growth factor (EGF)-like peptide supplementation during IVM improve subsequent oocyte developmental outcomes. This study investigated the effects of an IVM system that incorporates these two concepts. Cumulus-oocyte complexes (COCs) were collected from pre-pubertal mice either 46 hr post-equine chorionic gonadotropin (eCG) (IVM) or post-eCG + post-human chorionic gonadotropin (hCG) stimulation (in vivo maturation; IVV). IVM COCs were treated with the cAMP modulators forskolin and IBMX for 1, 2, or 4 hr (pre-IVM phase) prior to IVM. COCs then underwent IVM with the EGF-like peptides amphiregulin or epiregulin, or with the common IVM stimulants follicle-stimulating hormone (FSH) or EGF. A pre-IVM phase increased the size of the subsequent blastocysts' inner-cell-mass compared to standard IVM, regardless of IVM treatment (P < 0.05). Unlike FSH or EGF, amphiregulin or epiregulin significantly increased blastocyst quality (trophectoderm and total cell numbers) and/or yield (P < 0.01) compared to standard IVM, and were the only treatments that produced blastocysts comparable to IVV-derived blastocysts. Forskolin acutely up-regulated EGF-like peptide mRNA expression after a 2-hr pre-IVM phase (P < 0.001), although EGF receptor and ERK1/2 activities were not significantly different than control. IVV-like levels of EGF-like peptide mRNA expression during IVM were maintained only by supplementing with EGF-like peptides and EGF, since expression levels induced by FSH were significantly lower in vitro than during IVV. However, EGF receptor and ERK1/2 phosphorylation levels were not significantly different across treatment groups. In conclusion, a pre-IVM phase in conjunction with IVM in the presence of EGF-like peptides endows high oocyte developmental competence, as evidenced by increased embryo yield and/or quality relative to FSH and EGF.

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development.

PURPOSE: We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. METHODS: Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. RESULTS: No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21% vs 48%; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. CONCLUSIONS: This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.

Pioneering contributions by Robert Edwards to oocyte in vitro maturation (IVM).

The history of in vitro maturation (IVM) of mammalian oocytes, especially of human oocytes, holds a special place for Robert Edwards. He was the first to comprehensively examine and demonstrate maturation of human oocytes in vitro and in so doing he changed the course of medicine by fertilizing them in vitro. In reviewing his contribution, we have examined the state of the field at the time and discuss his pioneering insights into mammalian oocyte biology. We will also discuss how some of the major concepts and challenges identified by Edwards 50 years ago remain among the major challenges facing IVM today.

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation.

The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen-thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7-24 h from activation) by 2',7'-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.

Mode of oocyte maturation affects EGF-like peptide function and oocyte competence.

The function and impact of epidermal growth factor (EGF)-like peptide signalling during ovulation and in vivo oocyte maturation (IVV) has been recently characterized, however, little is currently known about the effect of oocyte in vitro maturation (IVM) on this pathway. The aim of this study was to examine expression and functional aspects of three EGF-like peptides (amphiregulin, epiregulin and betacellulin) and their common receptor (EGFR) in cumulus cells during mouse oocyte IVM compared with IVV. Cumulus-oocyte complexes (COCs) were collected from prepubertal mice either 46 h post-eCG (IVM) or 46 h post-eCG plus 0.5-12 h post-hCG (IVV). Time course experiments showed mRNA expression of all three EGF-like peptides and amphiregulin protein in IVM media were significantly lower for the majority of FSH-supplemented IVM compared with IVV. The supplementation of EGF during IVM yielded EGF-like peptide expression levels comparable with IVV and amphiregulin/epiregulin supplemented IVM. However, despite this, EGF activation of the COC EGFR remained significantly lower at 3 and 6 h of IVM than in vivo, and levels were similar to those observed during FSH-supplemented IVM. The addition of exogenous epiregulin during IVM significantly increased blastocyst rates, and epiregulin and amphiregulin improved blastocyst quality, compared with FSH or EGF. In conclusion, findings from this study suggest that the widely used IVM additives, FSH and EGF, are inadequate propagators of the essential EGF-like peptide signalling cascade. In contrast, the use of epiregulin and/or amphiregulin during IVM leads to improved oocyte developmental competence and therefore may be preferable IVM additives than FSH or EGF.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence.

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Estimation of glucose uptake by ovarian follicular cells.

In vitro maturation (IVM) of mammalian oocytes provides an alternative to traditional in vitro fertilization techniques for clinical treatment of infertility or animal breeding. IVM involves the collection of oocytes from the ovary prior to ovulation, with maturation occurring in a laboratory environment. The success of IVM is highly sensitive to the in vitro nutrient environment. The nurse cells surrounding the oocyte, known as cumulus cells, regulate this environment and removal of these cells reduces the ability of the oocyte to develop following insemination. Determining the nature of the interaction between the oocyte and cumulus cells, collectively called the cumulus-oocyte complex (COC), is a difficult task experimentally. Here we use a combination of experimental and mathematical techniques to investigate glucose transport within bovine COCs and find quantitative estimates of the glucose uptake rates of the oocyte and cumulus cells. Surprisingly, our modeling shows the rate of uptake of glucose by the oocyte to increase and then decrease with concentration, a result that needs further experimental investigation but which supports the expectation that high and low glucose concentrations are detrimental to oocyte development. The methodology described is suitable for use across species and for investigating the transport of other important nutrients within the COC.

Spray desorption collection of free fatty acids onto a solid phase microextraction fiber for trap grease analysis in biofuel production.

Spray desorption collection (SDC) is a sample collection and preparation method that allows for the collection of soluble chemical compounds directly from solid surfaces. Here the analysis of trap grease, a potential biofuel feedstock, is demonstrated by combining SDC with solid phase micro-extraction (SPME) collection followed by direct GC-MS analysis. The SPME fiber collects droplets of solvent, which has picked up analytes from the solid sample surface. It is found that the SDC-SPME combination is a simple and convenient method to collect sample components from surfaces when they are less volatile than heptanoic acid, while the collection efficiency of highly volatile compounds is increasingly reduced due to the purging effect of the nebulising gas. In a real trap grease analysis the SDC-SPME method was able to analyze both the longer chain fatty acids in the sample, important for energy production evaluation of the sample, as well as volatile sample components down to pentanoic acid, which may add to off-odours produced during biofuel use.

Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes.

BACKGROUND: Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system. METHODS AND RESULTS: Bovine or mouse cumulus-oocyte complexes (COCs) were treated with cAMP modulators for the first 1-2 h in vitro (pre-IVM), increasing COC cAMP levels ∼100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55%; SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF). CONCLUSIONS: SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.