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Does the ketone acetoacetate (AcAc) alone, or combined with β-hydroxybutyrate (βOHB), impact mouse embryo development, metabolism, histone acetylation and viability?Pronucleate mouse oocytes were cultured in vitro in G1/G2 media supplemented with ketones (AcAc or AcAc + βOHB) at concentrations representing those in maternal serum during pregnancy (0.04 mmol/l AcAc, 0.1 mmol/l βOHB), standard diet consumption (0.1 mmol/l AcAc, 0.25 mmol/l βOHB), ketogenic diet consumption (0.8 mmol/l AcAc, 2 mmol/l βOHB) and diabetic ketoacidosis (2 mmol/l AcAc, 4 mmol/l βOHB). Day 5 blastocysts were assessed for cell allocation, glucose metabolism and histone acetylation. Day 4 blastocysts exposed to 0.8 mmol/l AcAc + 2 mmol/l βOHB were transferred to standard-fed recipient females, and E14.5 fetal and placental development assessed.Exposure to 2 mmol/l AcAc or 0.8 mmol/l AcAc + 2 mmol/l βOHB did not impair blastocyst development, but significantly increased glucose consumption (P = 0.001 each), lowered glycolytic flux (P = 0.01, P 0.001) and elevated trophectoderm (TE) histone 3 lysine 27 acetylation (H3K27ac; P 0.001 each) compared with unexposed controls. Preimplantation AcAc + βOHB exposure reduced post-implantation fetal development by 25% (P = 0.037), and delayed female-specific fetal limb development (P = 0.019) and estimated fetal age (P = 0.019) compared with controls.Preimplantation exposure to ketones affects underlying metabolism and histone acetylation in blastocysts that are associated with persistent, female-specific perturbations in fetal development. A periconceptional diet that elevates ketone concentrations may impair human embryonic viability.

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.

The process of implantation is characterized by a complex cross-talk between the endometrium and the blastocyst, with the endometrium only being receptive to implantation during a transient window of implantation of approximately 2-3 days during the midsecretory phase. The timing of embryo transfer, including frozen embryo transfer, is therefore critical to the success of implantation. In this article, we discuss various elements that may guide the timing of frozen embryo transfer, including the role of endometrial characteristics such as thickness, days postovulation or length of progesterone administration, stage of the embryo, and the application of endometrial receptivity tests to guide personalized embryo transfer.

SummaryThe transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical mitochondrial (mt)DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics.We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived mtDNA by trophoblast cells of murine embryos, which associated with a reduction in mitochondrial respiration and ATP production.These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that mtDNA becomes packaged into secreted EVs that the embryo uptakes, and that the transfer of mtDNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

What is the effect on mouse fetal gene expression of combined antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and alpha-lipoic acid; A3) when used in culture media and vitrification/warming solutions?A laboratory-based analysis of an animal model. Embryo transfers were conducted on in-vivo-flushed blastocysts, or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14.5 fetal liver and placental tissue in all groups were quantified using RNA-Seq and functional analyses (gene ontology [GO] biological processes and Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway analysis).Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression. Notably, supplementation of in-vitro culture media or vitrification/warming solutions with A3 reduced the number of differentially expressed genes (DEG) and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly within the E14.5 placenta. Specifically, A3 supplementation significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction, along with genes involved in metabolism, cell senescence and cancer associated pathways. However, despite these improvements, several biological processes remained over-represented following both in-vitro culture and vitrification, even in the presence of A3.Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression, with the number of DEG greater following vitrification. Supplementation with A3 reduced the number of DEG and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly in the placenta. Notably, A3 supplementation of in-vitro culture media significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction.

To address the proposition that 'the way to improve ART outcomes is through the introduction of more technologies in the laboratory', it is prudent to first define what is considered to be improved outcomes. Evidently, this equates to an increase in the live birth rate but it should also include parameters such as time to pregnancy, cumulative pregnancy per oocyte retrieval and health of the resultant child. Furthermore, being able to maintain clinical results week in, week out through quality management also contributes to the overall success of a clinic, and hence can be considered an improved outcome. With regards to these outcomes, it is offered that not only does the introduction of several new technologies (defined here as instrumentation, techniques and enhanced computer utilization and analysis) have the potential to improve outcomes, but also some of them have the capacity to facilitate automation and standardization in the ART laboratory. Although the automation of procedures can be perceived as a justifiable goal itself, in this contribution the emphasis is on how new technologies could help more patients become parents of healthy children in the shortest possible time.

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 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 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.

OBJECTIVES: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division (‘fast’) develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos (‘slow’). Further, metabolomics analysis employing LC–MS conducted on groups of ‘fast’ blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. METHODS: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. RESULTS: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual ‘fast’ blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In ‘fast’ blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from ‘fast’ blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from ‘slow’ blastocysts. CONCLUSIONS: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Over the past four decades our ability to maintain a viable human embryo in vitro has improved dramatically, leading to higher implantation rates. This has led to a notable shift to single blastocyst transfer and the ensuing elimination of high order multiple gestations. Future improvements to embryo culture systems will not only come from new improved innovative media formulations (such as the inclusion the addition of antioxidants), but plausibly by moving away from static culture to more dynamic perfusion-based systems now made a reality due to breakthroughs in 3D printing technology and micro fabrication. Such an approach has already made it feasible to create high resolution devices for ICSI, culture and cryopreservation, paving the way not only for improvements in outcomes but also automation of ART. While improvements in culture systems can lead to further increases in pregnancy outcomes, the ability to quantitate biomarkers of embryo health and viability will reduce time to pregnancy and decrease pregnancy loss. Currently artificial intelligence is being used to assess embryo development through image analysis, but we predict its power will be realised through the creation of selection algorithms based on the integration of information related to metabolic functions, cell free DNA, morphokinetics, thereby using vast amounts of different data types obtained for each embryo to predict outcomes. All of this will not only make ART more effective, but it will also make it more cost effective, thereby increasing patient access to infertility treatment worldwide.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to placental hyperplasia in mammalian offspring generated by somatic cell nuclear transfer (SCNT). In this study, we show that offspring from mouse oocytes lacking the Polycomb protein Embryonic Ectoderm Development (EED) were initially growth restricted, characterised by low blastocyst cell counts and substantial mid-gestational developmental delay. This initial developmental delay was followed by striking late-gestational placental hyperplasia, fetal catch-up growth and extended gestational length that culminated in offspring overgrowth. This involved remodelling of the placenta, including expansion of fetal and maternal tissues and conspicuous expansion of the glycogen enriched cell population in the junctional zone that was associated with a delay in parturition. Despite this remodelling and offspring catchup growth, fetal/placental weight ratio and fetal blood glucose levels were low indicating low placental efficiency. Genome-wide analyses identified extensive transcriptional dysregulation in affected placentas, including a range of imprinted and non-imprinted genes and increased expression of the H3K27me3-imprinted geneSlc38a4,which regulates transport of essential amino acids in the placenta. Our data provide an explanation for apparently opposing observations of growth restriction and overgrowth of offspring derived fromEed-nulloocytes and demonstrate that PRC2-dependent programming in the oocyte regulates fetal and placental growth and developmental outcomes.

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 germlines.

STUDY QUESTIONWhat is the effect of the ketone β-hydroxybutyrate (βOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability?SUMMARY ANSWERIn vitro βOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner.WHAT IS KNOWN ALREADYA maternal KD in humans elevates gamete and offspring βOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to βOHB at supraphysiological concentrations (8–80 mM) perturbs preimplantation mouse embryo development.STUDY DESIGN, SIZE, DURATIONA mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with βOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured βOHB-exposed embryos was assessed.PARTICIPANTS/MATERIALS, SETTING, METHODSMouse embryos were cultured in vitro with or without βOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of βOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, βOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 β-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed.MAIN RESULTS AND THE ROLE OF CHANCEAll βOHB concentrations tested slowed embryo development (P LIMITATIONS, REASONS FOR CAUTIONThis study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro βOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of βOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown.WIDER IMPLICATIONS OF THE FINDINGSThese findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to βOHB within a physiological range. Maternal diets which increase βOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of βOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

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

STUDY QUESTIONIs there a relationship between blastocyst metabolism and biomarkers of embryo viability?SUMMARY ANSWERBlastocysts with higher developmental potential and a higher probability of resulting in a viable pregnancy consume higher levels of glucose and exhibit distinct amino acid profiles.WHAT IS KNOWN ALREADYMorphological and morphokinetic analyses utilized in embryo selection provide insight into developmental potential, but alone are unable to provide a direct measure of embryo physiology and inherent health. Glucose uptake is a physiological biomarker of viability and amino acid utilization is different between embryos of varying qualities.STUDY DESIGN, SIZE, DURATIONTwo hundred and nine human preimplantation embryos from 50 patients were cultured in a time-lapse incubator system in both freeze all and fresh transfer cycles. A retrospective analysis of morphokinetics, morphology (Gardner grade), KIDScore, artificial intelligence grade (EmbryoScore), glucose and amino acid metabolism, and clinical pregnancies was conducted.PARTICIPANTS/MATERIALS, SETTING, METHODSICSI was conducted in all patients, who were aged ≤37 years and previously had no more than two IVF cycles. Embryos were individually cultured in a time-lapse incubator system, and those reaching the blastocyst stage had their morphokinetics annotated and were each assigned a Gardner grade, KIDScore and EmbryoScore. Glucose and amino acid metabolism were measured. Clinical pregnancies were confirmed by the presence of a fetal heartbeat at 6 weeks of gestation.MAIN RESULTS AND THE ROLE OF CHANCEGlucose consumption was at least 40% higher in blastocysts deemed of high developmental potential using either the Gardner grade (P LIMITATIONS, REASONS FOR CAUTIONSamples were collected from patients who had ICSI treatment and from only one clinic.WIDER IMPLICATIONS OF THE FINDINGSThese results confirm that metabolites, such as glucose and amino acids, are valid biomarkers of embryo viability and could therefore be used in conjunction with other systems to aid in the selection of a healthy embryo.STUDY FUNDING/COMPETING INTEREST(S)Work was supported by Virtus Health. D.K.G is contracted with Virtus Health. The other authors have no conflict of interest to declare.TRIAL REGISTRATION NUMBERN/A.

text Globally, 39% of the adult population is overweight or obese, with the prevalence of obesity following an upward trajectory over the recent decades (WHO). Up to 30% of women of reproductive age in Western countries are obese before conception, and obese women experience higher rates of infertility and pregnancy complications than lean women; however, the mechanisms underpinning obesity-related infertility are poorly understood. Advanced Glycation Endproducts (AGEs) are a proinflammatory modification of proteins exposed to sugars, formed through the Maillard reaction. AGEs are elevated four-fold in the uterine fluid of obese, infertile women, compared to lean. AGEs equimolar to those in the obese microenvironment negatively impact the functions of endometrial epithelial and stromal cells, and adhesion and invasion of trophoblast cells, reducing the potential for successful maternal-fetal interactions (Antoniotti et al., 2018). This research further investigated preimplantation embryo development and endometrial cell functions in the presence of AGEs equimolar to those in obese uterine fluid. Altered local environments in very early life can set offspring up for a lifetime of health or disease (DoHAD); thus, uterine AGEs may contribute to the prevalence of non-communicable disease in children of obese parents. Preimplantation mouse embryos were cultured in vitro with AGEs equimolar with uterine fluid concentrations from lean and obese women, and their development and implantation potential assessed. “Obese” AGEs did not impact the proportion of embryos reaching blastocyst stage by day 4, but significantly reduced the proportion of blastocysts hatching by day 5 (P < 0.01). AGEs equimolar with the obese uterine environment detrimentally impacted trophectoderm formation and function: reduced trophectoderm cell number (P < 0.01), reduced outgrowth on fibronectin (indicative of reduced implantation potential, (P < 0.01), but did not increase cell apoptosis (TUNEL assay). RAGE antagonism, but neither metformin nor antioxidants, improved trophectoderm cell number. Thus, obesity-associated AGEs link obesity and reduced fertility through poor placentation potential of embryos (Hutchison et al, 2020). Endometrial epithelial cell function was examined in the presence of lean and obese concentrations of AGEs. Obese AGEs significantly reduced the rate of proliferation (xCelligence real time cell analysis) of the endometrial epithelial cell line ECC-1 versus lean AGEs (P = 0.04). Antioxidants successfully restored the rate of proliferation in the presence of obese AGEs (P = 0.7 versus lean AGEs). Subsequently, human endometrial epithelial organoid culture was utilised as a more physiologically relevant experimental paradigm. When cultured as organoids, primary endometrial epithelial cells were functionally responsive to obesity-associated AGEs, expressing both RAGE and TLR4. The morphology of organoids in culture was not impacted by the presence of obese AGEs versus lean; however, the proliferation of epithelial cells retrieved from organoid culture was altered by obese AGEs versus lean. Obese AGEs also increased the secretion of proinflammatory CXCL16 versus vehicle control (P = 0.04) while increased secretion of other proinflammatory cytokines and chemokines including TNFa approached significance in the presence of obese AGEs. As the inflammatory milieu is altered in the uterine fluid of infertile women, elevated AGEs may promote an infertile endometrial inflammatory environment. AGEs link obesity and reduced fertility, being detrimental to preimplantation embryo development and endometrial cell function when present at concentrations equal to those in obese uterine fluid. Antioxidants and RAGE antagonism provide beneficial effects to cell function in the presence of obesity-associated AGEs. This research provides evidence supporting AGEs as a factor contributing to obesity related infertility, and as an emerging frontier for reproductive health. Clinically, reduction of uterine AGEs may improve fertility for obese couples wishing to conceive. Antoniotti et al (2018). Hum Rep. 33(4), 654-665. PMID: 29471449 Hutchison et al (2020). RBMO. 41(5), 757-766. PMID: 32972872

Human induced pluripotent stem cells (iPSCs) can be differentiated in vitro into bona fide cardiomyocytes for disease modelling and personalized medicine. Mitochondrial morphology and metabolism change dramatically as iPSCs differentiate into mesodermal cardiac lineages. Inhibiting mitochondrial fission has been shown to promote cardiac differentiation of iPSCs. However, the effect of hydrazone M1, a small molecule that promotes mitochondrial fusion, on cardiac mesodermal commitment of human iPSCs is unknown. Here, we demonstrate that treatment with M1 promoted mitochondrial fusion in human iPSCs. Treatment of iPSCs with M1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. The pro-fusion and pro-cardiogenic effects of M1 were not associated with changes in expression of the α and β subunits of adenosine triphosphate (ATP) synthase. Our findings demonstrate for the first time that hydrazone M1 is capable of promoting cardiac differentiation of human iPSCs, highlighting the important role of mitochondrial dynamics in cardiac mesoderm lineage specification and cardiac development. M1 and other mitochondrial fusion promoters emerge as promising molecular targets to generate lineages of the heart from human iPSCs for patient-specific regenerative medicine.

The transition to pluripotency invokes profound metabolic restructuring; however, reprogramming is accompanied by the retention of somatic cell metabolic and epigenetic memory. Modulation of metabolism during reprogramming has been shown to improve reprogramming efficiency, yet it is not known how metabolite availability during reprogramming affects the physiology of resultant induced pluripotent stem cells (iPSCs). Metabolic analyses of iPSCs generated under either physiological (5%; P-iPSC) or atmospheric (20%; A-iPSC) oxygen conditions revealed that they retained aspects of somatic cell metabolic memory and failed to regulate carbohydrate metabolism with A-iPSC acquiring different metabolic characteristics. A-iPSC exhibited a higher mitochondrial membrane potential and were unable to modulate oxidative metabolism in response to oxygen challenge, contrasting with P-iPSC. RNA-seq analysis highlighted that A-iPSC displayed transcriptomic instability and a reduction in telomere length. Consequently, inappropriate modulation of metabolism by atmospheric oxygen during reprogramming significantly impacts the resultant A-iPSC metabolic and transcriptional landscape. Furthermore, retention of partial somatic metabolic memory in P-iPSC derived under physiological oxygen suggests that metabolic reprogramming remains incomplete. As the metabolome is a regulator of the epigenome, these observed perturbations of iPSC metabolism will plausibly have downstream effects on cellular function and physiology, both during and following differentiation, and highlight the need to optimize nutrient availability during the reprogramming process. Stem Cells 2019;37:1042–1056

STUDY QUESTION Can a deep learning model predict the probability of pregnancy with fetal heart (FH) from time-lapse videos? SUMMARY ANSWER We created a deep learning model named IVY, which was an objective and fully automated system that predicts the probability of FH pregnancy directly from raw time-lapse videos without the need for any manual morphokinetic annotation or blastocyst morphology assessment. WHAT IS KNOWN ALREADY The contribution of time-lapse imaging in effective embryo selection is promising. Existing algorithms for the analysis of time-lapse imaging are based on morphology and morphokinetic parameters that require subjective human annotation and thus have intrinsic inter-reader and intra-reader variability. Deep learning offers promise for the automation and standardization of embryo selection. STUDY DESIGN, SIZE, DURATION A retrospective analysis of time-lapse videos and clinical outcomes of 10 638 embryos from eight different IVF clinics, across four different countries, between January 2014 and December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS The deep learning model was trained using time-lapse videos with known FH pregnancy outcome to perform a binary classification task of predicting the probability of pregnancy with FH given time-lapse video sequence. The predictive power of the model was measured using the average area under the curve (AUC) of the receiver operating characteristic curve over 5-fold stratified cross-validation. MAIN RESULTS AND THE ROLE OF CHANCE The deep learning model was able to predict FH pregnancy from time-lapse videos with an AUC of 0.93 [95% CI 0.92–0.94] in 5-fold stratified cross-validation. A hold-out validation test across eight laboratories showed that the AUC was reproducible, ranging from 0.95 to 0.90 across different laboratories with different culture and laboratory processes. LIMITATIONS, REASONS FOR CAUTION This study is a retrospective analysis demonstrating that the deep learning model has a high level of predictability of the likelihood that an embryo will implant. The clinical impacts of these findings are still uncertain. Further studies, including prospective randomized controlled trials, are required to evaluate the clinical significance of this deep learning model. The time-lapse videos collected for training and validation are Day 5 embryos; hence, additional adjustment would need to be made for the model to be used in the context of Day 3 transfer. WIDER IMPLICATIONS OF THE FINDINGS The high predictive value for embryo implantation obtained by the deep learning model may improve the effectiveness of previous approaches used for time-lapse imaging in embryo selection. This may improve the prioritization of the most viable embryo for a single embryo transfer. The deep learning model may also prove to be useful in providing the optimal order for subsequent transfers of cryopreserved embryos. STUDY FUNDING/COMPETING INTEREST(S) D.T. is the co-owner of Harrison AI that has patented this methodology in association with Virtus Health. P.I. is a shareholder in Virtus Health. S.C., P.I. and D.G. are all either employees or contracted with Virtus Health. D.G. has received grant support from Vitrolife, the manufacturer of the Embryoscope time-lapse imaging used in this study. The equipment and time for this study have been jointly provided by Harrison AI and Virtus Health.

RESEARCH QUESTION Is the blastocyst's idiosyncratic metabolic production of lactate, and creation of a specialized microenvironment at the implatation site, an important mediator of maternal-fetal signalling to promote endometrial receptivity and implantation? DESIGN Hormonally primed ECC-1 and Ishikawa cells were used to assess functional changes to the endometrial epithelium after exposure to lactic acid (LA), LA with neutralized pH (nLA) or acidic pH (pHL). Tight junction integrity (transepithelial resistance [TER]), cellular proliferation or changes to gene expression by RT-PCR were analysed. The effect of LA on Endometrial stromal cells decidualization and migratory capacity, and HUVEC endothelial tube formation and angiogenesis, were also assessed. RESULTS Treatment of ECC-1 cells with 2.5 mM (P = 0.0037), 5 mM (P = 0.0044), 7.5 mM and 10 mM (P = 0.003) (P = 0.0021) LA significantly decreased the rate of cellular proliferation while TER was decreased with exposure to 2.5 mM LA (P = 0.024), 5 mM LA (P = 0.021) and 7.5 mM LA (P = 0.033). Exposure to nLA or pHL had no effect on proliferation or TER. Upregulation of GLUT4 (P = 0.002), GPR81 (P = 0.048), VEGF, SNAI1 (both P < 0.001) and RELA (P = 0.023) mRNA expression was observed after exposure of Ishikawa cells to combined LA plus pHL. Lactic acid increased the migratory capacity of decidualized stromal cells (P = 0.047) without changing the extent of decidualization. HUVEC tube formation was significantly increased by 5 mM LA exposure (P = 0.009). CONCLUSIONS The identification of LA as an important mediator in the maternal-fetal dialogue underpinning implantation is supported. Further examination of the role of LA within the infertile or compromised endometrium could improve natural and assisted pregnancy success and needs further investigation.

The aim of this article is to gather 9 thought leaders and their team members to present their ideas about the future of in vitro fertilization and the andrology laboratory. Although we have seen much progress and innovation in the laboratory over the years, there is still much to come, and this article looks at what these leaders think will be important in the future development of technology and processes in the laboratory.

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 LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION In this proof of principle study, the effects of V-SPs on embryonic development were only analysed in a mouse model. WIDER IMPLICATIONS OF THE FINDINGS These findings suggest that SPs represent a novel method by which a targeted dose of therapeutic agents (e.g. bioactive VEGF) can be delivered to the developing in vitro embryo to promote embryonic development, an approach that negates the breakdown of VEGF associated with storage in solution. As such, V-SPs may be an alternative and effective method of delivering bioactive VEGF to the developing in vitro embryo; however, the potential use of V-SPs in clinical IVF requires further investigation. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the University of Melbourne. The authors have no conflict of interest to declare.

The in vitro fertilization and andrology laboratories are at the center of assisted reproductive technologies and the place where technicians and embryologists manipulate gametes and preimplantation-stage embryos with the goal of achieving the best embryo for transfer. Through the years, these laboratories have seen developments in technique, technology, and testing. The goal of this Views and Interviews series is to bring together the thought leaders in the field and envision what the laboratories will look like in the next 10 years.