A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Optical imaging detects metabolic signatures associated with oocyte quality†.

Oocyte developmental potential is intimately linked to metabolism. Existing approaches to measure metabolism in the cumulus oocyte complex (COC) do not provide information on the separate cumulus and oocyte compartments. Development of an assay that achieves this may lead to an accurate diagnostic for oocyte quality. Optical imaging of the autofluorescent cofactors reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides a spatially resolved indicator of metabolism via the optical redox ratio (FAD/[NAD(P)H + FAD]). This may provide an assessment of oocyte quality. Here, we determined whether the optical redox ratio is a robust methodology for measuring metabolism in the cumulus and oocyte compartments compared with oxygen consumption in the whole COC. We also determined whether optical imaging could detect metabolic differences associated with poor oocyte quality (etomoxir-treated). We used confocal microscopy to measure NAD(P)H and FAD, and extracellular flux to measure oxygen consumption. The optical redox ratio accurately reflected metabolism in the oocyte compartment when compared with oxygen consumption (whole COC). Etomoxir-treated COCs showed significantly lower levels of NAD(P)H and FAD compared to control. We further validated this approach using hyperspectral imaging, which is clinically compatible due to its low energy dose. This confirmed lower NAD(P)H and FAD in etomoxir-treated COCs. When comparing hyperspectral imaged vs non-imaged COCs, subsequent preimplantation development and post-transfer viability were comparable. Collectively, these results demonstrate that label-free optical imaging of metabolic cofactors is a safe and sensitive assay for measuring metabolism and has potential to assess oocyte developmental competence.

Fabrication on the microscale: a two-photon polymerized device for oocyte microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. METHODS: The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-µm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). RESULTS: Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. CONCLUSION: This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.

The effect of discrete wavelengths of visible light on the developing murine embryo.

PURPOSE: A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. METHODS: Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. RESULTS: Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. CONCLUSION: Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device.

PURPOSE: Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. METHODS: The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). RESULTS: Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. CONCLUSION: The Pod and Garage system minimized the volume of cryoprotectant at vitrification-by ~ 1000-fold-improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

HYPOXIA AND REPRODUCTIVE HEALTH: Hypoxia and ovarian function: follicle development, ovulation, oocyte maturation.

The ovarian follicle provides the oocyte with the ideal environment for growth and development in preparation for ovulation and fertilisation. The follicle undergoes many structural changes as it grows, including changes in vasculature, cell proliferation and differentiation and the formation of a fluid-filled antrum. These changes collectively create a low oxygen environment within the follicle. Thus, the oocyte itself develops in a potentially hypoxic environment. The survival of hypoxic tissues is controlled by hypoxia-inducible factors (HIFs) that are activated in a low oxygen state. The understanding of HIF pathways is growing across all fields of biology, and its role in ovarian development is steadily gaining clarity. One of the genes upregulated by HIF is a vascular endothelial growth factor, the main inducer of angiogenesis which is required for follicle development and corpus formation. Ovulation is also intrinsically linked to HIF activity through the ovulatory luteinising hormone surge increasing HIF expression. The role for HIF in oocyte maturation is less understood, as efforts to replicate the low oxygen environment of the in vivo follicle are not achievable by culturing in low oxygen alone. There is potential for other factors present in vivo, but lost in vitro, to be involved in oxygen regulation. One factor of interest is haemoglobin, the oxygen-binding protein, which brings the exciting possibility of sensitive oxygen regulation, consequently affecting HIF-regulated gene expression. A thorough understanding of oxygen regulation within the follicle would provide vital applications for the field of assisted reproductive technologies, in particular in vitro oocyte maturation.

Effect of oxygen and glucose availability during in vitro maturation of bovine oocytes on development and gene expression.

PURPOSE: Oxygen tension during the in vitro maturation (IVM) of oocytes is important for oocyte developmental competence. A conflict exists in the literature as to whether low oxygen during IVM is detrimental or beneficial to the oocyte. Many research and clinical labs use higher than physiological oxygen tension perhaps believing that low-oxygen tension is detrimental to oocyte development. Other studies show that glucose is important if low-oxygen tension is used during maturation. In this study, we look at the link between low oxygen and glucose availability during IVM to resolve misconceptions around low-oxygen tension during IVM. METHODS: Bovine cumulus oocyte complexes (COCs) were matured at 20% vs 7% oxygen in media containing differing glucose concentrations or varying availability. Cleavage and blastocyst rates were recorded. RT-PCR determined expression levels of metabolic, oxygen, and stress-responsive genes following IVM. RESULTS: Embryo development in 7% oxygen groups with 2.3mM glucose/low glucose availability was lower than 20% oxygen groups. Under 7% oxygen with 5.6mM glucose or higher glucose availability, rates were restored to those seen in 20% oxygen. Expressions of BNIP3, ENO1, GAPDH, and SLC2A1, were upregulated in 7% oxygen/low glucose, compared to 20% oxygen groups. BNIP3 expression was higher in 7% oxygen group with low glucose availability compared to the 20% groups. CONCLUSION: Oocyte developmental competence is negatively impacted following IVM in low oxygen when glucose availability is limited. Glucose concentration and physical culture conditions need to be considered when comparing the effects of different oxygen concentrations during IVM.

Dysregulation of bisphosphoglycerate mutase during in vitro maturation of oocytes.

PURPOSE: Oxygen is vital for oocyte maturation; however, oxygen regulation within ovarian follicles is not fully understood. Hemoglobin is abundant within the in vivo matured oocyte, indicating potential function as an oxygen regulator. However, hemoglobin is significantly reduced following in vitro maturation (IVM). The molecule 2,3-bisphosphoglycerate (2,3-BPG) is essential in red blood cells, facilitating release of oxygen from hemoglobin. Towards understanding the role of 2,3-BPG in the oocyte, we characterized gene expression and protein abundance of bisphosphoglycerate mutase (Bpgm), which synthesizes 2,3-BPG, and whether this is altered under low oxygen or hemoglobin addition during IVM. METHODS: Hemoglobin and Bpgm expression within in vivo matured human cumulus cells and mouse cumulus-oocyte complexes (COCs) were evaluated to determine physiological levels of Bpgm. During IVM, Bpgm gene expression and protein abundance were analyzed in the presence or absence of low oxygen (2% and 5% oxygen) or exogenous hemoglobin. RESULTS: The expression of Bpgm was significantly lower than hemoglobin when mouse COCs were matured in vivo. Following IVM at 20% oxygen, Bpgm gene expression and protein abundance were significantly higher compared to in vivo. At 2% oxygen, Bpgm was significantly higher compared to 20% oxygen, while exogenous hemoglobin resulted in significantly lower Bpgm in the COC. CONCLUSION: Hemoglobin and 2,3-BPG may play a role within the maturing COC. This study shows that IVM increases Bpgm within COCs compared to in vivo. Decreasing oxygen concentration and the addition of hemoglobin altered Bpgm, albeit not to levels observed in vivo.

Time-lapse confocal imaging-induced calcium ion discharge from the cumulus-oocyte complex at the time of cattle oocyte activation.

Oocyte activation, the dynamic transformation of an oocyte into an embryo, is largely driven by Ca2+ oscillations that vary in duration and amplitude across species. Previous studies have analysed intraoocyte Ca2+ oscillations in the absence of the oocyte's supporting cumulus cells. Therefore, it is unknown whether cumulus cells also produce an ionic signal that reflects fertilisation success. Time-lapse confocal microscopy and image analysis on abattoir-derived cattle cumulus-oocyte complexes coincubated with spermatozoa revealed a distinct discharge of fluorescence from the cumulus vestment. This study demonstrated that this Ca2+ fluorescence discharge was an artefact induced by the imaging procedure independently of oocyte activation success. The fluorescence discharge was a direct result of cumulus cell membrane integrity loss, and future studies should consider the long-term effect of fluorescent labels on cells in time-lapse imaging. However, this study also demonstrated that the distinctive pattern of a coordinated fluorescence discharge was associated with both the presence of spermatozoa and subsequent embryo development to the morula stage, which was affected by Ca2+ chelation and a reduction in the active efflux of the fluorophore. This indicates that the cumulus vestment may have a relationship with oocyte activation at and beyond fertilisation that requires further investigation.

Conditions to optimise the developmental competence of immature equine oocytes.

Optimising the developmental potential of immature equine oocytes and invitro-produced (IVP) embryos was explored through modifications of established media and holding temperature. In Experiment 1, delaying spontaneous resumption of meiosis through the process of simulated physiological oocyte maturation with the addition of the adenylate cyclase activator forskolin (50µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100µM) to overnight holding medium before maturation improved blastocyst production (P<0.05). In Experiment 2, the blastocyst production rate was increased significantly when cumulin (100ng mL-1) was added to the overnight holding or culture media (P<0.05). In Experiment 3, immature oocytes held overnight at 16°C before maturation had improved developmental competence than those held at 20°C and 5°C (P<0.05). There was no difference between maturation rates, but blastocyst formation per cleaved oocyte was significantly greater in oocytes held overnight at 16°C than at 20°C or 5°C. Furthermore, blastocyst formation per recovered oocyte and per fertilised oocyte was greater when oocytes were held before maturation at 16°C than at 5°C (P<0.05). In Experiment 4, the addition of sodium ascorbate (AC; 50µg mL-1) to the maturation and/or culture media of oocytes and IVP embryos did not improve blastocyst production, but did appear to lower cleavage rates compared with oocytes and embryos cultured without AC.

Hemoglobin: potential roles in the oocyte and early embryo†.

Hemoglobin (Hb) is commonly known for its capacity to bind and transport oxygen and carbon dioxide in erythroid cells. However, it plays additional roles in cellular function and health due to its capacity to bind other gases including nitric oxide. Further, Hb acts as a potent antioxidant, quenching reactive oxygen species. Despite its potential roles in cellular function, the preponderance of Hb research remains focused on its role in oxygen regulation. There is increasing evidence that Hb expression is more ubiquitous than previously thought, with Hb and its variants found in a myriad of cell types ranging from macrophages to spermatozoa. The majority of nonerythroid cell types that express Hb are situated within hypoxic environments, suggesting Hb may play a role in hypoxia-inducible factor-regulated gene expression by controlling the level of oxygen available or as an adaptation to low oxygen providing a mechanism to store oxygen. Oocyte maturation and preimplantation embryo development occur within the low oxygen environments of the antral follicle and oviduct/uterus, respectively. Interestingly, Hb was recently found in human cumulus and granulosa cells and murine cumulus-oocyte complexes and preimplantation embryos. Here, we consolidate and analyze the research generated todate on Hb expression in nonerythroid cells with a particular focus on reproductive cell types. We outline future directions of this research to elucidate the role of Hb during oocyte maturation and preimplantation embryo development and finally, we explore the potential clinical applications and benefits of Hb supplementation during the in vitro culture of gametes and embryos.

The effect of streptozotocin-induced hyperglycemia on N-and O-linked protein glycosylation in mouse ovary.

Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography-ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans; with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

STUDY QUESTION: Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? SUMMARY ANSWER: Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. WHAT IS KNOWN ALREADY: Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. STUDY DESIGN, SIZE, DURATION: The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. PARTICIPANTS/MATERIALS, SETTING, METHODS: In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. LIMITATIONS, REASONS FOR CAUTION: Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. WIDER IMPLICATIONS OF THE FINDINGS: Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). 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. The authors declare there are no conflict of interest.

Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation.

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes.

Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 µM), oleic acid (200 µM), and steric acid (75 µM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

The definition of IVM is clear-variations need defining.

Oocyte in vitro maturation (IVM) is currently defined as the maturation in vitro of immature cumulus-oocyte complexes collected from antral follicles. This is the original definition as first described by Pincus and Enzmann and then by Edwards many decades ago, and this clear and unambiguous definition has served us well ever since. In an attempt to clarify apparent differences among clinicians, the following revised definition of IVM was recently proposed: 'The retrieval of oocytes from small and intermediate sized follicles in an ovary before the largest follicle has surpassed 13 mm in mean diameter'. As such, this proposed definition should encompass the use of hCG triggering. To change the clear and long-serving definition of IVM to fit varying clinical practices requires a compelling justification based on solid scientific and clinical grounds. We are of the opinion that the proposed revised definition of IVM is counterintuitive as it includes protocols that are intended to mature oocytes in vivo The proposed definitions are cumbersome and indeed further complicate the situation. It is not scientifically rational to base the definition on follicular size, and the definition ignores the vast corporate knowledge acquired from the many decades and >6000 publications in animal research that universally practices IVM as per the existing definition. Furthermore, such a definition can lead to false results in interpreting the follow-up of children conceived using IVM. Hence, we see no rationale to change the existing definition of IVM. However, we agree that variations on IVM require alternative nomenclature-these definitions need to be intuitive and need to clearly distinguish themselves from the existing long-standing definition of IVM. This would help to clarify the recent confusion within the clinical ART community as to what is and what is not, IVM.

The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts.

The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon-tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4 mM) with or without exposure to DNP (0, 10, 100 µM) between Days 5 and 8 post-fertilization. The absence or presence of high (4 mM) glucose reduced blastocyst development and favored the development of male embryos (P < 0.001). DNP at 10 µM had no effect, whereas 100 µM had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10 µM DNP further skewed the sex ratio toward males (P < 0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (<2.5 mM) medium to minimize potential embryonic stress, as higher concentrations have sexually dimorphic effects on development and an embryo's ability to signal to the maternal reproductive tract.

Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult individual; indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc.