A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

In brief: A ketogenic diet (KD) elevates blood ß-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. Abstract: A ketogenic diet elevates blood ß-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters ß-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and ß-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and ß-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated ß-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Temperature fluctuations during embryo transfer can be mitigated by optimizing transfer protocol.

RESEARCH QUESTION: What impact do variations in embryo transfer catheter loading and movement procedures have on temperature and pH fluctuations during embryo transfer? DESIGN: Mock embryo transfers were conducted to test the impact of air flow/movement, use of catheter coverings, and the type of workstation used for catheter loading on catheter temperature. A thermocouple probe inserted into the tip of the outer catheter or taped to the exterior of the inner catheter recorded temperature within the catheter every 5 s from time of mock embryo loading (TL) to 60 s (TL + 60 s) or from the start of transit (TT). Fluctuations in culture medium pH in embryo transfer dishes were monitored. RESULTS: The rate of cooling during transit was faster (all P < 0.05) when catheters were uncovered compared with all covering methods tested. This resulted in a lower catheter temperature at TL + 20 s (28.43 ± 0.30 °C) compared with catheters covered by plastic tubing (31.4 ± 0.30 °C), paper (31.0 ± 0.26 °C) or paper + thumb (31.1 ± 0.78 °C; all P ≤ 0.05). Temperature was maintained more effectively when catheters were loaded in a crib compared with a heated stage, until initiation of transit, when the rate of temperature decrease was similar. Culture medium pH increased more rapidly when embryo transfer dishes remained on a heated stage during the procedure compared with in an open crib. CONCLUSIONS: Temperature loss during the embryo transfer procedure can be mitigated by reducing the transit time and using catheter coverings. Use of a crib for catheter loading only improved temperature stability while the catheter remained in the crib, not during transit, and reduced pH fluctuations during the procedure.

Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

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.

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.

A step closer to parenthood with non-obstructive azoospermia: Unveiling the impact of microdissection testicular sperm extraction in Australia's largest single-centre study.

BACKGROUND: Non-obstructive azoospermia (NOA) diagnosis poses challenges for couples seeking parenthood. Microdissection testicular sperm extraction (MD-TESE) excels in retrieving testicular sperm cells for NOA cases. However, limited live birth data in Australian NOA patients hinders accurate counselling. AIMS: This study aimed to determine the likelihood of infertile couples with a male partner diagnosed with NOA conceiving biological children using MD-TESE / intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: A retrospective cohort study included 108 NOA men treated at a public fertility unit and a private fertility centre (May 2009-May 2022). PRIMARY OUTCOME: live birth rate (LBR); secondary outcomes: sperm retrieval rate, pregnancy rate, and neonatal outcomes. RESULTS: Among 108 patients undergoing MD-TESE, the positive sperm retrieval rate (PSRR) was 64.8% (70/108). Histology best predicted sperm retrieval success, with hypo-spermatogenesis yielding a 94.1% PSRR. Age, testicular volume, and hormonal parameters had no significant impact. Mean male age: 35.4 years; mean partner age: 32.7 years. Fertilisation rate: 50.7%. LBR per initiated cycle: 58.7% (37/63); per embryo transfer: 63.8% (37/58); per initially diagnosed NOA man: 34.3% (37/108). Cumulative LBR: 74.1% (43/58); twin rate: 10.8% (4/37). No neonatal deaths or defects were observed among 47 live offspring. CONCLUSION: This study provides valuable data for counselling NOA couples on the probability of conceiving biological offspring. MD-TESE and ICSI yielded favourable PSRR (64.8%) and LBR (63.8%). However, couples should be aware that once NOA is confirmed, the chance of taking home a baby is 34%.

Acetoacetate and ß-hydroxybutyrate reduce mouse embryo viability via differential metabolic and epigenetic mechanisms.

RESEARCH QUESTION: Does the ketone acetoacetate (AcAc) alone, or combined with ß-hydroxybutyrate (ßOHB), impact mouse embryo development, metabolism, histone acetylation and viability? DESIGN: Pronucleate mouse oocytes were cultured in vitro in G1/G2 media supplemented with ketones (AcAc or AcAc + ßOHB) at concentrations representing those in maternal serum during pregnancy (0.04 mmol/l AcAc, 0.1 mmol/l ßOHB), standard diet consumption (0.1 mmol/l AcAc, 0.25 mmol/l ßOHB), ketogenic diet consumption (0.8 mmol/l AcAc, 2 mmol/l ßOHB) and diabetic ketoacidosis (2 mmol/l AcAc, 4 mmol/l ßOHB). 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 ßOHB were transferred to standard-fed recipient females, and E14.5 fetal and placental development assessed. RESULTS: Exposure to 2 mmol/l AcAc or 0.8 mmol/l AcAc + 2 mmol/l ßOHB 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 + ßOHB 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. CONCLUSION: 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.

Preimplantation embryo exposure to ketone bodies exerts sex-specific effects on mouse fetal and placental transcriptomes.

RESEARCH QUESTION: Does in vitro exposure of preimplantation mouse embryos to the ketone bodies ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc) impact post-transfer fetal and placental gene expression? DESIGN: Blastocysts cultured in vitro with or without 2 mmol/l ßOHB alone ('ßOHB') or combined with 0.8 mmol/l AcAc ('Keto') underwent embryo transfer. Transcriptional profiles of sexed placenta, liver and brain at gestational day 14.5 were examined via RNA sequencing and DAVID functional analysis. RESULTS: A sexually dimorphic response to in vitro ketone exposure was observed. Both ßOHB and Keto exposure down-regulated genes related to oxidative phosphorylation specifically in female liver. ßOHB down-regulated female placental steroid biosynthetic processes, while Keto treatment up-regulated genes relevant to blood vessel formation and cell migration in male placenta. Brain transcriptomes were minimally affected. X-linked genes and chromatin modifiers were identified as differentially expressed in both liver and placenta, alluding to a sex-specific regulatory mechanism. CONCLUSIONS: Transient preimplantation ketone exposure perturbs sex-specific fetal liver and placental gene expression, demonstrating a developmental programming effect that warrants future investigation of the postnatal metabolic health of male and female offspring.

Detrimental actions of obesity-associated advanced glycation end-products on endometrial epithelial cell proliferation are alleviated by antioxidants.

RESEARCH QUESTION: Advanced glycation end-products (AGE) are elevated in the uterine environment of obese infertile women. Can the detrimental effects of AGE on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)? DESIGN: Human endometrial epithelial cells (ECC-1) were exposed to AGE at concentrations physiologically representative of uterine fluid in lean or obese individuals, and three potential therapeutics: 25 nmol/l receptor for AGE (RAGE) antagonist FPS-ZM1, 100 µmol/l metformin, or a combination of antioxidants (10 µmol/l N-acetyl-l-cysteine, 10 µmol/l N-acetyl-l-carnitine and 5 µmol/l α-lipoic acid). Real-time cell analysis (xCELLigence, ACEA Biosciences) determined the rate of adhesion and proliferation. The proliferation of organoid-derived cells and secretion of cytokines from organoids was characterized in the presence of AGE (n = 5). The uterine fluid of women undergoing assisted reproduction was profiled for AGE-associated inflammatory markers (n = 77). RESULTS: ECC-1 proliferation was reduced by AGE from obese versus lean conditions and vehicle control (P = 0.04 and P < 0.001, respectively), and restored to a proliferation corresponding to lean conditions by antioxidants. AGE influenced organoid derived primary endometrial epithelial cell proliferation in a donor-dependent manner. AGE increased the organoid secretion of the proinflammatory cytokine CXCL16 (P = 0.006). Clinically, CXCL16 correlated positively to maternal body mass index (R = 0.264, P = 0.021) and intrauterine glucose concentration (R = 0.736, P < 0.0001). CONCLUSIONS: Physiologically relevant concentrations of AGE alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGE-treated endometrial epithelial (ECC-1) cells. Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and CXCL16 secretion in the presence of AGE equimolar with the uterine fluid from obese individuals.

ß-hydroxybutyrate reduces blastocyst viability via trophectoderm-mediated metabolic aberrations in mice.

STUDY QUESTION: What is the effect of the ketone ß-hydroxybutyrate (ßOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability? SUMMARY ANSWER: In vitro ßOHB 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 ALREADY: A maternal KD in humans elevates gamete and offspring ßOHB 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 ßOHB at supraphysiological concentrations (8-80 mM) perturbs preimplantation mouse embryo development. STUDY DESIGN, SIZE, DURATION: A mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with ßOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured ßOHB-exposed embryos was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos were cultured in vitro with or without ßOHB 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 ßOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, ßOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 ß-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All ßOHB concentrations tested slowed embryo development (P < 0.05), and ßOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, ßOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of ßOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25-4 mM) ßOHB elevated H3K27bhb levels. Preimplantation ßOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of ßOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of ßOHB was detected, whereby female fetuses from ßOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses. LIMITATIONS, REASONS FOR CAUTION: This study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro ßOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of ßOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to ßOHB within a physiological range. Maternal diets which increase ßOHB 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 ßOHB 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 NUMBER: N/A.

'The way to improve ART outcomes is to introduce more technologies in the laboratory'.

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.

Antioxidant supplementation of mouse embryo culture or vitrification media support more in-vivo-like gene expression post-transfer.

RESEARCH QUESTION: 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? DESIGN: 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). RESULTS: 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. CONCLUSION: 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.

A microenvironment of high lactate and low pH created by the blastocyst promotes endometrial receptivity and implantation.

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.

Post-warming embryo morphology is associated with live birth: a cohort study of single vitrified-warmed blastocyst transfer cycles.

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.

Mitochondrial and glycolytic remodeling during nascent neural differentiation of human pluripotent stem cells.

As human pluripotent stem cells (hPSCs) exit pluripotency, they reportedly switch from glycolytic energy production to primarily mitochondrial metabolism. Here, we show that upon ectoderm differentiation to neural precursor cells (NPCs), hPSCs increase glycolytic rate, ultimately producing more carbon as lactate than is consumed as glucose. However, glucose, lactate and pyruvate utilization decrease to half their PSC levels by the NPC stage, establishing a more quiescent metabolic state. Furthermore, we characterize a metabolic exit event within the first 24 h of differentiation, plausibly necessary to transition hPSCs out of the pluripotent state. Contrary to current thinking, mitochondrial mass does not increase during NPC induction. Instead, mitochondrial DNA copies and mitochondrial activity decrease, suggesting that mitochondrial metabolism either requires suppression, or is not required, for nascent ectoderm differentiation. Our work, therefore, contrasts with the dogma that the hPSC state is primarily glycolytic, transitioning to an oxidative metabolism upon the loss of the pluripotent state. Instead, we show that heightened glycolytic metabolism is acquired, indicating that metabolic modulation of both glycolysis and mitochondrial metabolism occurs during exit from pluripotency in hPSCs.

A pilot study investigating a novel particle-based growth factor delivery system for preimplantation embryo culture.

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 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.

Nicotinamide adenine dinucleotide induces a bivalent metabolism and maintains pluripotency in human embryonic stem cells.

Nicotinamide adenine dinucleotide (NAD+ ) and its precursor metabolites are emerging as important regulators of both cell metabolism and cell state. Interestingly, the role of NAD+ in human embryonic stem cell (hESC) metabolism and the regulation of pluripotent cell state is unresolved. Here we show that NAD+ simultaneously increases hESC mitochondrial oxidative metabolism and partially suppresses glycolysis and stimulates amino acid turnover, doubling the consumption of glutamine. Concurrent with this metabolic remodeling, NAD+ increases hESC pluripotent marker expression and proliferation, inhibits BMP4-induced differentiation and reduces global histone 3 lysine 27 trimethylation, plausibly inducing an intermediate naïve-to-primed bivalent metabolism and pluripotent state. Furthermore, maintenance of NAD+ recycling via malate aspartate shuttle activity is identified as an absolute requirement for hESC self-renewal, responsible for 80% of the oxidative capacity of hESC mitochondria. Our findings implicate NAD+ in the regulation of cell state, suggesting that the hESC pluripotent state is dependent upon cellular NAD+ .

A combination of growth factors and cytokines alter preimplantation mouse embryo development, foetal development and gene expression profiles.

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.

Antioxidants increase blastocyst cryosurvival and viability post-vitrification.

STUDY QUESTION: What is the effect of antioxidants acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid (A3) in vitrification and warming solutions on mouse blastocyst development and viability? SUMMARY ANSWER: The combination of three antioxidants in vitrification solutions resulted in mouse blastocysts with higher developmental potential in vitro and increased viability as assessed by both an outgrowth model in vitro and fetal development following uterine transfer. WHAT IS KNOWN ALREADY: The antioxidant combination of acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid present in IVF handling and embryo culture media has significant beneficial effects on mouse embryo and fetal development, especially under oxidative stress. STUDY DESIGN, SIZE, DURATION: The study was a laboratory-based analysis of an animal model. Rapid cooling through vitrification was conducted on F1 mouse blastocysts, with antioxidants (A3) supplemented in vitrification and/or warming solutions, followed by culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Pronucleate oocytes were collected and cultured in groups to Day 4 blastocysts. Expanded blastocysts were vitrified and warmed in solutions with and without the A3 antioxidants and cultured for a further 24 h. Blastocyst cell number and allocation, apoptosis and histone acetylation levels were all quantified, and viability through outgrowths and transfers assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse blastocysts vitrified with no antioxidants had significantly lower cell numbers (P  < 0.001) and higher apoptotic cells (P  < 0.05) compared to non-vitrified embryos. Addition of combined A3 antioxidants to the vitrification and warming solutions resulted in a significant increase in inner cell mass cell (ICM) number (P  < 0.001) and total cell number (P  < 0.01), and an increase in outgrowth area (P < 0.05), which correlated with the increased fetal weight (P < 0.05), crown rump length (P < 0.05) and limb development (P < 0.05) determined following transfer compared to embryos with no antioxidants. Furthermore, while blastocyst vitrification significantly reduced acetylation levels (P < 0.05) compared to non-vitrified embryos, the inclusion of A3 antioxidants helped to ameliorate this. LIMITATIONS, REASONS FOR CAUTION: Embryo development was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: Results in this study demonstrate that vitrification and warming of blastocysts have significant detrimental effects on embryo histone acetylation and subsequent viability. The presence of antioxidants in the vitrification solutions helps to alleviate the negative effects of cryopreservation. Our data indicate that antioxidants need to be present in the medium at the time of exposure to increased oxidative stress associated with vitrification and that prior exposure (i.e. during culture or IVF alone) is insufficient to protect cells against cryo-induced injury. Hence, A3 antioxidants may assist in maintaining the viability of vitrified human embryos in ART through the reduction of oxidative stress. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Metabolic activity of human blastocysts correlates with their morphokinetics, morphological grade, KIDScore and artificial intelligence ranking.

STUDY QUESTION: Is there a relationship between blastocyst metabolism and biomarkers of embryo viability? SUMMARY ANSWER: Blastocysts 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 ALREADY: Morphological 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, DURATION: Two 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, METHODS: ICSI 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 CHANCE: Glucose consumption was at least 40% higher in blastocysts deemed of high developmental potential using either the Gardner grade (P < 0.01, n = 209), KIDScore (P < 0.05, n = 207) or EmbryoScore (P < 0.05, n = 184), compared to less viable blastocysts and in blastocysts that resulted in a clinical pregnancy compared to those that failed to implant (P < 0.05, n = 37). Additionally, duration of cavitation was inversely related to glucose consumption (P < 0.05, n = 200). Total amino acid consumption was significantly higher in blastocysts with an EmbryoScore higher than the cohort median score (P < 0.01, n = 185). Furthermore, the production of amino acids was significantly lower in blastocysts with a high Gardner grade (P < 0.05, n = 209), KIDScore (P < 0.05, n = 207) and EmbryoScore (P < 0.01, n = 184). LIMITATIONS, REASONS FOR CAUTION: Samples were collected from patients who had ICSI treatment and from only one clinic. WIDER IMPLICATIONS OF THE FINDINGS: These 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 NUMBER: N/A.

Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming.

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.