Search

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30 × 10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

PURPOSE: This study aims to examine whether blastocyst morphology post-warming correlates with live birth. METHODS: In this cohort study, morphological characteristics post-warming were reviewed in all single vitrified-warmed blastocyst transfer cycles performed between November 2016 and May 2017. Immediately before transfer, the degree of blastocoel re-expansion was graded as A, fully expanded; B, partially expanded ≥ 50%; C, partially expanded < 50%; and D, collapsed. The degree of post-warming cell survival was graded on a scale of 50 to 100% and was then classified into 4 groups: very low 50-70%, low 71-80%, moderate 81-90%, and high 91-100%. RESULTS: Overall, 612 cycles were reviewed, of which 196 included PGT-A tested embryos. The live birth rate (LBR) increased from 11.4% in the collapsed blastocysts group to 38.9% in the post-warming full re-expansion group (p < 0.001) and from 6.5% for blastocysts with a very low cell survival rate to 34.7% for blastocysts with high cell survival rate (p = 0.001). LBR was 6.7% for blastocysts with the worst post-warming morphological characteristics, namely, collapsed with very low cell survival rate. On multivariate analyses, partial blastocyst re-expansion ≥ 50%, full re-expansion, and high cell survival rate remained significantly associated with live birth, after controlling for female age, pre-vitrification morphological grading, and PGT-A. A sub-analysis of cycles using PGT-A tested embryos showed similar results. CONCLUSION: Post-warming re-expansion and high cell survival rate are associated with higher LBR in euploid and untested blastocysts. However, embryos with poor post-warming morphology still demonstrate a considerable probability of live birth, and they should not be discarded.

BACKGROUND: Non-genetic disease inheritance and offspring phenotype are substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2-dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. RESULTS: In this study, we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. CONCLUSIONS: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes and that this activity is conserved in the mouse and human

STUDY QUESTION: Can vascular endothelial growth factor (VEGF)-loaded silica supraparticles (V-SPs) be used as a novel mode of delivering VEGF to the developing preimplantation embryo in vitro? SUMMARY ANSWER: Supplementation of embryo culture media with V-SPs promoted embryonic development in a manner equivalent to media supplemented with free VEGF. WHAT IS KNOWN ALREADY: VEGF is a maternally derived growth factor that promotes preimplantation embryonic development in vitro. However, its use in clinical media has limitations due to its low stability in solution. STUDY DESIGN, SIZE, DURATION: This study was a laboratory-based analysis utilising a mouse model. V-SPs were prepared in vitro and supplemented to embryonic culture media. The bioactivity of V-SPs was determined by analysis of blastocyst developmental outcomes (blastocyst development rate and total cell number). PARTICIPANTS/MATERIALS, SETTING, METHODS: SPs were loaded with fluorescently labelled VEGF and release kinetics were characterised. Bioactivity of unlabelled VEGF released from V-SPs was determined by analysis of embryo developmental outcomes (blastocyst developmental rate and total cell number) following individual mouse embryo culture in 20 µl of G1/G2 media at 5% oxygen, supplemented with 10 ng/ml recombinant mouse VEGF in solution or with V-SPs. The bioactivity of freeze-dried V-SPs was also assessed to determine the efficacy of cryostorage. MAIN RESULTS AND THE ROLE OF CHANCE: VEGF release kinetics were characterised by an initial burst of VEGF from loaded spheres followed by a consistent lower level of VEGF release over 48 h. VEGF released from V-SPs resulted in significant increases in total blastocyst cell number relative to the control (P < 0.001), replicating the effects of medium freely supplemented with fresh VEGF (P < 0.001). Similarly, freeze dried V-SPs exerted comparable effects on embryonic development (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this proof

Within the maternal tract, the preimplantation embryo is exposed to an array of growth factors (GFs) and cytokines, most of which are absent from culture media used in clinical IVF. Whilst the addition of individual GFs and cytokines to embryo culture media can improve preimplantation mouse embryo development, there is a lack of evidence on the combined synergistic effects of GFs and cytokines on embryo development and further foetal growth. Therefore, in this study, the effect of a combined group of GFs and cytokines on mouse preimplantation embryo development and subsequent foetal development and gene expression profiles was investigated. Supplementation of embryo culture media with an optimised combination of GFs and cytokines (0.05 ng/ml vascular endothelial GF, 1 ng/ml platelet-derived GF, 0.13 ng/ml insulin-like GF 1, 0.026 ng/ml insulin-like GF 2 and 1 ng/ml granulocyte colony-stimulating factor) had no effect on embryo morphokinetics but significantly increased trophectoderm cell number (P = 0.0002) and total cell number (P = 0.024). Treatment with this combination of GFs and cytokines also significantly increased blastocyst outgrowth area (P < 0.05) and, following embryo transfer, increased foetal weight (P = 0.027), crown-rump length (P = 0.017) and overall morphological development (P = 0.027). RNA-seq analysis of in vitro derived foetuses identified concurrent alterations to the transcriptional profiles of liver and placental tissues compared with those developed in vivo, with greater changes observed in the GF and cytokine treated group. Together these data highlight the importance of balancing the actions of such factors for the regulation of normal development and emphasise the need for further studies investigating this prior to clinical implementation.

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.

Reprogramming to pluripotency involves drastic restructuring of both metabolism and the epigenome. However, induced pluripotent stem cells (iPSC) retain transcriptional memory, epigenetic memory, and metabolic memory from their somatic cells of origin and acquire aberrant characteristics distinct from either other pluripotent cells or parental cells, reflecting incomplete reprogramming. As a critical link between the microenvironment and regulation of the epigenome, nutrient availability likely plays a significant role in the retention of somatic cell memory by iPSC. Significantly, relative nutrient availability impacts iPSC reprogramming efficiency, epigenetic regulation and cell fate, and differentially alters their ability to respond to physiological stimuli. The significance of metabolites during the reprogramming process is central to further elucidating how iPSC retain somatic cell characteristics and optimising culture conditions to generate iPSC with physiological phenotypes to ensure their reliable use in basic research and clinical applications. This review serves to integrate studies on iPSC reprogramming, memory retention and metabolism, and identifies areas in which current knowledge is limited.

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

BACKGROUND: Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. RESULTS: Here we provide evidence that Polycomb Repressive Complex 2 (PRC2) regulates paternal inheritance. Reduced PRC2 function in mice resulted in male sub-fertility and altered epigenetic and transcriptional control of retrotransposed elements in foetal male germ cells. Males with reduced PRC2 function produced offspring that over-expressed retrotransposed pseudogenes and had altered preimplantation embryo cleavage rates and cell cycle control. CONCLUSION: This study reveals a novel role for the histone-modifying complex, PRC2, in paternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance.

Reprogramming somatic cells to a pluripotent cell state (induced Pluripotent Stem (iPS) cells) requires reprogramming of metabolism to support cell proliferation and pluripotency, most notably changes in carbohydrate turnover that reflect a shift from oxidative to glycolytic metabolism. Some aspects of iPS cell metabolism differ from embryonic stem (ES) cells, which may reflect a parental cell memory, or be a consequence of the reprogramming process. In this study, we compared the metabolism of 3 human iPS cell lines to assess the fidelity of metabolic reprogramming. When challenged with reduced oxygen concentration, ES cells have been shown to modulate carbohydrate use in a predictably way. In the same model, 2 of 3 iPS cell lines failed to regulate carbohydrate metabolism. Oxygen is a well-characterized regulator of cell function and embryo viability, and an inability of iPS cells to modulate metabolism in response to oxygen may indicate poor metabolic fidelity. As metabolism is linked to the regulation of the epigenome, assessment of metabolic responses of iPS cells to physiological stimuli during characterization is warranted to ensure complete cell reprogramming and as a measure of cell quality.

OBJECTIVE: In vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos. METHODS: Mouse embryos were cultured individually in 2 µL of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6. RESULTS: Mouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p<0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control (101.95±3.36 vs. 91.31±3.33, p<0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells (79.86±3.29, p<0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control (22.9%±1.1%, 23.3%±1.1%, and 23.1%±1.1% vs. 19.5%±1.0%, p<0.05). CONCLUSION: These data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.

Spiny mice of the genus Acomys display several unique physiological traits, including menstruation and scar-free wound healing; characteristics that are exceedingly rare in mammals, and of considerable interest to the scientific community. These unique attributes, and the potential for spiny mice to accurately model human diseases, are driving increased use of this genus in biomedical research, however little genetic information is accessible for this species. This project aimed to generate a draft transcriptome for the Common spiny mouse (Acomys cahirinus). Illumina sequencing of RNA from 15 organ types (male and female) produced 451 million, 150 bp paired-end reads (92.4Gbp). An extensive survey of de novo transcriptome assembly approaches using Trinity, SOAPdenovo-Trans, and Oases at multiple kmer lengths was conducted, producing 50 single-kmer assemblies from this dataset. Non-redundant transcripts from all assemblies were merged into a meta-assembly using the EvidentialGene tr2aacds pipeline, producing the largest gene catalogue to date for Acomys cahirinus. This study provides the first detailed characterization of the spiny mouse transcriptome. It validates use of the EvidentialGene tr2aacds pipeline in mammals to augment conventional de novo assembly approaches, and provides a valuable scientific resource for further investigation into the unique physiological characteristics inherent in the genus Acomys.

STUDY QUESTION: Can a generally applicable morphokinetic algorithm suitable for Day 3 transfers of time-lapse monitored embryos originating from different culture conditions and fertilization methods be developed for the purpose of supporting the embryologist's decision on which embryo to transfer back to the patient in assisted reproduction? SUMMARY ANSWER: The algorithm presented here can be used independently of culture conditions and fertilization method and provides predictive power not surpassed by other published algorithms for ranking embryos according to their blastocyst formation potential. WHAT IS KNOWN ALREADY: Generally applicable algorithms have so far been developed only for predicting blastocyst formation. A number of clinics have reported validated implantation prediction algorithms, which have been developed based on clinic-specific culture conditions and clinical environment. However, a generally applicable embryo evaluation algorithm based on actual implantation outcome has not yet been reported. STUDY DESIGN, SIZE, DURATION: Retrospective evaluation of data extracted from a database of known implantation data (KID) originating from 3275 embryos transferred on Day 3 conducted in 24 clinics between 2009 and 2014. The data represented different culture conditions (reduced and ambient oxygen with various culture medium strategies) and fertilization methods (IVF, ICSI). The capability to predict blastocyst formation was evaluated on an independent set of morphokinetic data from 11 218 embryos which had been cultured to Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: The algorithm was developed by applying automated recursive partitioning to a large number of annotation types and derived equations, progressing to a five-fold cross-validation test of the complete data set and a validation test of different incubation conditions and fertilization methods. The results were expressed as receiver operating characteristics curves using the area under the c

The effect of a short-term feeding and starvation experiment on juvenile abalone (Haliotis rubra × H. laevigata) was investigated (average length = 67 mm; average weight = 48 g). All aquaculture experiments were conducted at The University of Melbourne, Australia. Artificial feed was supplied ad libitum to the fed group, and no feed was supplied to the starved group. A modified metabolite extraction protocol using deuterated solvents was developed for ¹H-NMR-based metabolite profiling of digestive gland in response to the short-term feeding/starvation experiment, to avoid lyophilisation prior to biochemical analysis. PLS-DA revealed that fed and starved abalone are metabolically distinct from each other after 28 and 56 days. After 28 days, the fed group was defined by an increase in arginine, glucose, glutamate, glycine, inosine and uracil (P < 0.05), and the starved group was defined by an increase in N,N-dimethylglycine. After 56 days, the fed group still displayed increased glucose (P < 0.05), while N,N-dimethylglycine remained elevated in the starved group (P < 0.05). Arginine and glycogen were all higher at 28 days compared to 56 days, suggesting decreased anaerobic energy production at the later time point. Only glucose and N,N-dimethylglycine were significantly different between the fed and starved groups after 56 days, suggesting that abalone had not acclimatised to the starvation treatment after 28 days. These results infer N,N-dimethylglycine is a robust marker for short-term starvation in abalone. ¹H-NMR was also conducted on the artificial feed and starved abalone faecal matter, revealing the biochemical differences between them and digestive gland tissue. These methodology and results will facilitate a deeper understanding of the nutritional and physiological requirements of abalone in an aquaculture setting.

The differentiation of pluripotent stem cells is associated with extensive changes in metabolism, as well as widespread remodeling of the epigenetic landscape. Epigenetic regulation is essential for the modulation of differentiation, being responsible for cell type specific gene expression patterns through the modification of DNA and histones, thereby establishing cell identity. Each cell type has its own idiosyncratic pattern regarding the use of specific metabolic pathways. Rather than simply being perceived as a means of generating ATP and building blocks for cell growth and division, cellular metabolism can directly influence cellular regulation and the epigenome. Consequently, the significance of nutrients and metabolites as regulators of differentiation is central to understanding how cells interact with their immediate environment. This review serves to integrate studies on pluripotent stem cell metabolism, and the regulation of DNA methylation and acetylation and identifies areas in which current knowledge is limited.

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen provides important mediators that modulate the endometrium and support the conceptus during implantation. Previously we demonstrated the importance of vascular endothelial growth factor (VEGF) in the human uterus, particularly with respect to embryo implantation. In the current study, proteomic analysis of human uterine lavage fluid identified the presence of placental growth factor (PlGF) a homolog of VEGF, that binds the VEGF receptor 1 (VEGFR1). Analysis of immunostaining for PlGF in human endometrial tissue across the menstrual cycle (from both fertile and infertile women) revealed PlGF was predominantly localised to glandular and luminal epithelial cells, with staining in the decidualising stromal cells surrounding the maternal spiral arteries in the secretory phase of the menstrual cycle. Immunoreactive PlGF was also detected in subpopulations of endometrial leukocytes. Functional studies demonstrated that culturing mouse embryos with recombinant human (rh)PlGF enhanced blastocyst cell number and outgrowth. Furthermore, treatment of human endometrial epithelial cells (EEC) with rhPlGF enhanced EEC adhesion. Taken together, these data demonstrate that PlGF is abundant in the human endometrium, and secreted into the uterine lumen where it mediates functional changes in cellular adhesion with important roles in implantation.

Oxygen is a powerful regulator of cell function and embryonic development. It has previously been determined that oxygen regulates human embryonic stem (hES) cell glycolytic and amino acid metabolism, but the effects on mitochondria are as yet unknown. Two hES cell lines (MEL1, MEL2) were analyzed to determine the role of 5% (physiological) and 20% (atmospheric) oxygen in regulating mitochondrial activity. In response to extended physiological oxygen culture, MEL2 hES cells displayed reduced mtDNA content, mitochondrial mass and expression of metabolic genes TFAM, NRF1, PPARa and MT-ND4. Furthermore, MEL2 hES cell glucose consumption, lactate production and amino acid turnover were elevated under physiological oxygen. In stark contrast, MEL1 hES cell amino acid and carbohydrate use and mitochondrial function were relatively unaltered in response to oxygen. Furthermore, differentiation kinetics were delayed in the MEL1 hES cell line following BMP4 treatment. Here we report the first incidence of metabolic dysfunction in a hES cell population, defined as a failure to respond to oxygen concentration through the modulation of metabolism, demonstrating that hES cells can be perturbed during culture despite exhibiting the defining characteristics of pluripotent cells. Collectively, these data reveal a central role for oxygen in the regulation of hES cell metabolism and mitochondrial function, whereby physiological oxygen promotes glucose flux and suppresses mitochondrial biogenesis and gene expression.

Embryo transfer is a commonly performed surgical technique. In mice, protocols typically specify pairing recipient females with vasectomized males to induce a receptive uterine environment for embryo implantation. However, this induced receptive state is not always maintained until implantation occurs. The use of a well-characterized correlation between oestrous state and exfoliative vaginal cytology was therefore evaluated to assess uterine receptivity immediately before embryo transfer. Eight- to 12-week-old virgin female CD1 mice (n = 22) were paired overnight with vasectomized males and successfully mated, indicated by the presence of a vaginal plug. These dams underwent embryo transfer 3 days later with embryos obtained from superovulated 4-week-old F1 (C57BL/6 × CBA) females. Non-invasive vaginal lavage was conducted immediately before transfer. Dams were killed 6 days after transfer and the uterus collected for histological analysis. Embryo implantation rate in mice was 96% when cytological analysis of the lavage samples signified dioestrus (n = 6), whereas the implantation rate was <15% (n = 16) when cytology signified other stages of oestrous. This simple, quick, non-invasive measure of receptivity was accurate and easily adopted and, when applied prospectively, will avoid unnecessary surgery and subsequent culling of non-suitable recipients, while maximizing the implantation potential of each recipient female.

Fetal growth restriction (FGR) is a major obstetric complication stemming from poor placental development. We have previously demonstrated that paternal obesity in mice is associated with impaired embryo development and significantly reduced fetal and placental weights. We hypothesised that the FGR observed in our rodent model of paternal diet-induced obesity is associated with alterations in metabolic, cell signalling and stress pathways. Male C57BL/6 mice were fed either a normal or high-fat diet for 10 weeks before sperm collection for IVF and subsequent embryo transfer. On embryonic day 14, placentas were collected and RNA extracted from both male and female placentas to assess mRNA expression of 24 target genes using custom RT-qPCR arrays. Peroxisome proliferator-activated receptor alpha (Ppara) and caspase-12 (Casp12) expression were significantly altered in male placentas from obese fathers compared with normal (P<0.05), but not female placentas. PPARA and CASP12 proteins were localised within the placenta to trophoblast giant cells by immunohistochemistry, and relative protein abundance was determined by western blot analysis. DNA was also extracted from the same placentas to determine methylation status. Global DNA methylation was significantly increased in female placentas from obese fathers compared with normal (P<0.05), but not male placentas. In this study, we demonstrate for the first time that paternal obesity is associated with changes in gene expression and methylation status of extraembryonic tissue in a sex-specific manner. These findings reinforce the negative consequences of paternal obesity before conception, and emphasise the need for more lifestyle advice for prospective fathers.

STUDY QUESTION: Does combined parental obesity, both an obese mother and father, have a greater effect on mouse preimplantation embryo development and quality than single-parent obesity? SUMMARY ANSWER: Combined parental obesity causes a greater reduction in the blastocyst rate and a greater delay to the timing of key embryonic developmental events than single-parental obesity, as well as altering embryonic characteristics, such as zona pellucida width. WHAT IS KNOWN ALREADY: Maternal or paternal obesity alone are known to have significant and detrimental impacts on preimplantation embryo development. Furthermore, these early embryonic perturbations can have long-term impacts on both offspring health and further generations. This is one of the first studies to examine the effects of having both an obese mother and an obese father. STUDY DESIGN, SIZE, DURATION: A cross-sectional control versus treatment mouse study of diet-induced obesity was employed, in which 300 embryos per group were generated and studied from reciprocal matings: (i) control female and control male (Lean Parented Embryos); (ii) control female and obese male (Paternal Obese Parented Embryos); (iii) obese female and control male (Maternal Obese Parented Embryos) and (iv) obese female and obese male (Combined Obese Parented embryos). Assessments of the embryonic development rate, timing of development, morphological characteristics, metabolic gene expression, metabolism and cell lineage allocation were made at selected time points and analysed in relation to parental obesity status. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three-week-old C57BL6 male and female mice were fed control (7% total fat) or high fat (21% total fat) diets for a minimum of 8 weeks. Females were superovulated, mated, fertilized zygotes recovered and standard mouse in vitro embryo culture performed. Time-lapse monitoring was undertaken to compare developmental timings and morphological characteristics (embryonic area and zona

The mammalian blastocyst exhibits a high capacity for aerobic glycolysis, a metabolic characteristic of tumours. It has been considered that aerobic glycolysis is a means to ensure a high carbon flux to fulfil biosynthetic demands. Here, alternative explanations for this pattern of metabolism are considered. Lactate creates a microenvironment of low pH around the embryo to assist the disaggregation of uterine tissues to facilitate trophoblast invasion. Further it is proposed that lactate acts as a signalling molecule (especially at the reduced oxygen tension present at implantation) to elicit bioactive VEGF recruitment from uterine cells, to promote angiogenesis. Finally it is suggested that the region of high lactate/low pH created by the blastocyst modulates the activity of the local immune response, helping to create immune tolerance. Consequently, the mammalian blastocyst offers a model to study the role of microenvironments, and how metabolites and pH are used in signalling.

Low birth weight is associated with an increased risk for adult disease development with recent studies highlighting transmission to subsequent generations. However, the mechanisms and timing of programming of disease transmission to the next generation remain unknown. The aim of this study was to examine the effects of low birth weight and advanced maternal age on second-generation preimplantation blastocysts. Uteroplacental insufficiency or sham surgery was performed in late-gestation WKY pregnant rats, giving rise to first-generation (F1) restricted (born small) and control offspring respectively. F1 control and restricted females, at 4 or 12 months of age, were naturally mated with normal males. Second-generation (F2) blastocysts from restricted females displayed reduced expression of genes related to growth compared with F2 control (P<0.05). Following 24 h culture, F2 restricted blastocysts had accelerated development, with increased total cell number, a result of increased trophectoderm cells compared with control (P<0.05). There were alterations in carbohydrate and serine utilisation in F2 restricted blastocysts and F2 restricted outgrowths from 4-month-old females respectively (P<0.05). F2 blastocysts from aged restricted females were developmentally delayed at retrieval, with reduced total cell number attributable to reduced trophectoderm number with changes in carbohydrate utilisation (P<0.05). Advanced maternal age resulted in alterations in a number of amino acids in media obtained from F2 blastocyst outgrowths (P<0.05). These findings demonstrate that growth restriction and advanced maternal age can alter F2 preimplantation embryo physiology and the subsequent offspring growth.

Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also increased glucose uptake and changed amino acid turnover by the cells. These changes were distinct from those induced by serum and occurred in the absence of detectable differentiation. Induction of differentiation by bone morphogenetic protein 4 (BMP4), in contrast, did not alter metabolite turnover. Deviations from metabolite turnover by ES cells in fully defined medium demonstrated that culture environment can alter metabolite use. The challenge remains to understand the impact of metabolic changes on long-term cell maintenance and the functionality of derived cell populations.

Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring.