Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

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 growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

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.

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.

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.

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.

Advanced glycation end products present in the obese uterine environment compromise preimplantation embryo development.

RESEARCH QUESTION: Proinflammatory advanced glycation end products (AGE), highly elevated within the uterine cavity of obese women, compromise endometrial function. Do AGE also impact preimplantation embryo development and function? DESIGN: Mouse embryos were cultured in AGE equimolar to uterine fluid concentrations in lean (1-2 µmol/l) or obese (4-8 µmol/l) women. Differential nuclear staining identified cell allocation to inner cell mass (ICM) and trophectoderm (TE) (day 4 and 5 of culture). Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay (day 5). Day 4 embryos were placed on bovine serum albumin/fibronectin-coated plates and embryo outgrowth assessed 93 h later as a marker of implantation potential. AGE effects on cell lineage allocation were reassessed following pharmacological interventions: either 12.5 nmol/l AGE receptor (RAGE) antagonist; 0.1 nmol/l metformin; or combination of 10 µmol/l acetyl-l-carnitine, 10 µmol/l N-acetyl-l-cysteine, and 5 µmol/l alpha-lipoic acid. RESULTS: 8 µmol/l AGE reduced: hatching rates (day 5, P < 0.01); total cell number (days 4, 5, P < 0.01); TE cell number (day 5, P < 0.01), and embryo outgrowth (P < 0.01). RAGE antagonism improved day 5 TE cell number. CONCLUSIONS: AGE equimolar with the obese uterine environment detrimentally impact preimplantation embryo development. In natural cycles, prolonged exposure to AGE may developmentally compromise embryos, whereas following assisted reproductive technology cycles, placement of a high-quality embryo into an adverse 'high AGE' environment may impede implantation success. The modest impact of short-term RAGE antagonism on improving embryo outcomes indicates preconception AGE reduction via pharmacological or dietary intervention may improve reproductive outcomes for overweight/obese women.

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.

Culture of the Mouse Preimplantation Embryo.

The first culture media designed specifically to support development of the preimplantation mouse embryo were formulated over 50 years ago and were based on balanced salt solutions, containing the carbohydrates pyruvate, lactate, and glucose as the sole energy sources. Such media used a bicarbonate-carbon dioxide buffer system to maintain the desired pH, and were typically supplemented with serum albumin, but lacked free amino acids. In contrast to the complexity of a tissue culture medium, these original formulations of mouse embryo culture media were very simplistic. Over the intervening decades, as our understanding of the physiology and metabolism of the preimplantation embryo increased, together with a greater understanding of the environment within the female reproductive tract, culture media to support mouse embryo development in vitro have become more physiological and consequently more complex. A main addition to such media has been an array of amino acids. Although the media of today contain more components than their predecessors, their preparation remains relatively easy to accomplish, made feasible through the use of stock solutions, which also readily facilitates any changes to formulations to be made, an essential prerequisite for experimentation. As well as changes in media formulations, there have been exciting developments in incubator technology and design, such as the inclusion of time-lapse capability, redefining our ability to both culture and monitor embryo development in vitro.

Antioxidants improve mouse preimplantation embryo development and viability.

STUDY QUESTION: What is the effect of three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid), when used individually and in combination, on mouse embryo development in culture, and subsequent fetal development post-transfer? SUMMARY ANSWER: A combination of antioxidants resulted in significant increases in blastocyst cell number, maintained intracellular glutathione (GSH) levels, supported earlier cleavage times from 5-cell stage to expanded blastocyst, and improved fetal developmental irrespective of incubator oxygen concentration. WHAT IS KNOWN ALREADY: Acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid have been shown to have beneficial effects individually in several tissues, and most recently on developing embryos, in the presence of oxidative stress. STUDY DESIGN, SIZE, DURATION: Morphokinetics of mouse embryos were quantitated using time-lapse imaging. GSH levels in pronucleate oocytes were measured. Blastocysts underwent differential nuclear staining for inner cell mass and trophectoderm cells or were transferred to recipient females to assess implantation and fetal development. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Pronucleate oocytes from F1 mice were cultured in 5 or 20% oxygen either individually or in groups of 10, in media G1/G2, in the presence or absence of 10 µM acetyl-L-carnitine /10 µM N-acetyl-L-cysteine /5 µM α-lipoic acid, either individually or in combination. Controls were embryos cultured without antioxidants. Intracellular levels of reduced glutathione were quantitated in pronucleate oocytes. Embryo development and viability were analysed through time-lapse microscopy and embryo transfers. MAIN RESULTS AND THE ROLE OF CHANCE: Antioxidants significantly increased mouse blastocyst cell numbers compared with control when used individually (P< 0.05) and to a greater effect when all three were used in combination (P< 0.01) in 20% oxygen. The combination of antioxidants resulted in faster development rates to 5-cell cleavage stage, which continued until the expanded blastocyst stage when cultured in 20% oxygen. The beneficial effects of combining the antioxidants were greater for embryos cultured individually as opposed to in groups of 10 and for those embryos cultured in 20% compared to 5% oxygen. Levels of GSH were significantly decreased in control embryos that were incubated in the absence of antioxidants in 20% oxygen (P< 0.01), compared with in vivo flushed embryos. However, when embryos were cultured with antioxidants the level of GSH was not different to that of in vivo developed embryos. Embryos cultured in the presence of antioxidants in 20% oxygen and transferred resulted in significantly longer crown-rump length (11.6 ± 0.1 mm versus 11.3 ± 0.1 mm; P< 0.01), heavier fetuses (209.8 ± 11.8 mg versus 183.9 ± 5.9 mg; P< 0.05) and heavier placentas (103.5 ± 3.1 mg versus 93.6 ± 2.7 mg; P< 0.01) compared with controls (all data are mean ± SEM). Further, a post-implantation benefit of the antioxidant combination was also evident after culture in 5% oxygen. LIMITATIONS, REASONS FOR CAUTION: Embryo development and implantation was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: These findings show that a combination of antioxidants in the culture media has a highly beneficial effect on mouse preimplantation embryo development in vitro and on subsequent fetal development post-transfer. These data indicate a potential role for the inclusion of specific antioxidant combinations in human embryo culture media irrespective of oxygen concentration. However, before application to human embryos, a proper evaluation of this approach in prospective, preferably randomized, trials will be required. STUDY FUNDING/COMPETING INTERESTS: This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

[Cystectomy followed by cecocystoplasty: functional and oncologic results. An experience of 101 cases]. / Cystectomies suivies de caecocystoplasties: résultats fonctionnels et carcinologiques. Une expérience de 101 cas.

From 1979 to 1992, 101 radical cystectomy with caecoplasty were performed by the same operator (MR). Mean age of the patients was 62 years with a sex-ratio: 10 males/1 female. The indications were: Transitional Cell Carcinoma in 89 cases, neurologic bladder in 5 cases, interstitial cystitis in 3 cases tuberculosis bladder in 3 cases and lymphoma in one case. The operative mortality was 3% and postoperative morbidity was 8%. The surgical procedure consisted of performing a neo-bladder with ileocaecal segment. The technique of ureterocaecal implantation changed during the study period. On a functional point of view, 28 ureterocaecal stenosis were reported (15%). It were rare (2.4%) with the last ureterocaecal anastomosis technique. The diurnal continence rate was 100%. The nocturnal continence rate was only 25%. On uroflowmetric point of view, the detubularization permitted to obtain low pressure bladders with 15% rate of atonic bladder. Transverse taeniamyotomy of the caecum permitted to increase the uroflowmetric performance of the bladder without atonic risk. The 5 and 10 year overall actuarial survival rate were respectively 5% and 30%. Tumoral stage was a significant prognostic factor. An adjuvant chemotherapy was performed in 28 patients. The 5 year actuarial survival rate of the patients with a chemotherapy was 82% versus 28% for the patients without chemotherapy (p < 0.01). Caecocystoplasty after radical cystectomy was a reliable technique in our experience.