Effect of a novel copper chloride gel on endometrial growth and function in healthy volunteers.

RESEARCH QUESTION: Does the application of a micro-dose of copper chloride gel increase endometrial production of vascular endothelial growth factor (VEGF) without compromising endometrial function or producing embryo toxicity? DESIGN: An estimate of optimal dose was made based on cell culture studies. Ten healthy participants received an initial uterine application of placebo gel, followed by copper chloride gel (37.5 µM, 75 µM, or 150 µM dose) in a later hormone replacement cycle. Endometrial biopsies (day 5.5 luteal) and pelvic ultrasound were carried out during each cycle to evaluate endometrial function and growth. Uterine fluid was assessed for residual copper levels on the day of biopsy, and copper chloride gel underwent mouse embryos assay assessment for potential embryo toxicity. RESULTS: The copper gel significantly increased endometrial VEGF expression (quantitative polymerase chain reaction), and also increasing endometrial thickness by an average of 2.2 mm compared with matched control cycles. The copper gel did not adversely affect endometrial morphology or maturation (histological dating and molecular receptivity testing), and mouse embryos assay studies showed no evidence of embryo toxicity. Furthermore, uterine cavity flush samples mostly lacked copper, with only negligible amounts present in one sample. CONCLUSION: Applying copper chloride gel to the uterine cavity upregulated endometrial VEGF and significantly increased endometrial thickness and volume. No adverse effects on the endometrium or embryos were observed. Copper chloride gels show promise for treating suboptimal endometrial thickness if the results of this study are confirmed by larger randomized controlled trials.

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.

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.

Assessment and impact of paediatric internalized weight bias: A systematic review.

BACKGROUND: Internalized weight bias (IWB) refers to an individual's belief in negative weight-related stigma. Children and adolescents are particularly vulnerable to IWB, but little is known about IWB in this population. OBJECTIVE: To conduct a systematic review to (1) identify the instruments that measure IWB among children and adolescents and (2) explore comorbid variables associated with paediatric IWB. METHODS: This systematic review was conducted in accordance with the PRISMA guidelines. Articles were pulled from Ovid and PubMED Medline, Ovid HealthStar and ProQuest PsychInfo. Studies were included if they were observational studies, addressed the topic of IWB, and included children under the age of 18. Major outcomes were collected and analysed using inductive qualitative methods. RESULTS: 24 studies met inclusion/exclusion criteria. Researchers used two main instruments to measure IWB: Weight Bias Internalization Scale and Weight Self Stigma Questionnaire. There was some variation in the response scales and wording of these instruments between studies. Outcomes with significant associations were divided into four categories: physical health (n = 4), mental health (n = 9), social functioning (n = 5), and eating behaviours (n = 8). CONCLUSIONS: IWB is significantly associated with and may contribute to maladaptive eating behaviours and adverse psychopathology in children.

Associations of COVID-19 Lockdowns on Eating Behaviors and Body Mass Index in Patients with a History of Bariatric Surgery: a Cross-Sectional Analysis.

INTRODUCTION: Few studies have explored the impact of the COVID-19 pandemic on the eating behaviors, dietary quality, and changes in weight of postoperative bariatric surgery patients. METHODS: A cross-sectional survey on eating behaviors and attitudes toward food was emailed or given to patients who had bariatric surgery before March 2020. Patient charts were reviewed for weight measures. RESULTS: Seventy-five (71.43%) patients experienced weight recurrence with an average increase in body mass index (BMI) of 2.83 kg/m2 (SD: 2.19). The majority of patients reported no symptoms of binge eating (n = 81, 77.14%) with 16 (15.24%) qualifying for loss of control eating (LOCE). LOCE was significantly associated with grazing behavior (p = 0.04), emotional over-eating (p = 0.001), and food responsiveness (p = 0.002). LOCE was negatively associated with dietary quality (p = 0.0009) and satiety responsiveness (p = 0.01). Grazing behavior was significantly associated with emotional over-eating (p < 0.0001) and food responsiveness (p < 0.0001) as well as negatively associated with dietary quality (p < 0.0001). Slow eating was negatively associated with grazing (p = 0.01), emotional over-eating (p = 0.003), and food responsiveness (p < 0.0001). When included in a regression model controlling for age and sex, emotional over-eating was a significant predictor of weight recurrence (ß = 0.25; p = 0.04). CONCLUSION: Our results suggest that maladaptive eating behaviors contributed to LOCE and poor dietary quality during the COVID-19 pandemic; however, slow eating may be protective against grazing, emotional over-eating, and food responsiveness.

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.

Feline maxillary sarcoma: clinicopathologic features of spindle cell sarcomas from the maxilla of 25 cats.

CASE SERIES SUMMARY: Feline maxillary sarcomas are aggressive spindle cell neoplasms that occur within the maxilla, palate and upper lip of cats. This diagnosis includes fibrosarcoma and sarcomas with indeterminate histomorphology, excluding melanocytic tumors and sarcomas that can be differentiated by histomorphology. In this study of feline maxillary sarcomas in 25 cats, the cats' ages ranged from 4 to 16 years (median 12.5). These sarcomas presented as smooth thickenings or mass lesions of the gingiva and palate, often involving both the right and left quadrants of the maxilla. Radiographic bone loss was typically absent to mild at the time of diagnosis. Histologically, feline maxillary sarcomas were composed of spindle cells with varying amounts of fibrous stroma and mild inflammation. Metastasis was not documented for any cat in the study, although clinical staging was limited. Cats were often euthanized because of local recurrence following incomplete tumor excision and local tumor progression. Median survival time from the date of histologic diagnosis was 70 days (n = 12). RELEVANCE AND NOVEL INFORMATION: Feline maxillary sarcomas are aggressive neoplasms that may be difficult to differentiate from a benign, reactive process or other types of spindle cell neoplasms. Our findings indicate that feline maxillary sarcoma has distinctive clinical and histopathologic features, and the information provided in this paper will facilitate early and specific diagnosis of this tumor.

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.

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.

Substance Use and Overdose in Public Libraries: Results from a Five-State Survey in the US.

In the U.S., overdoses have become a health crisis in both public and private places. We describe the impact of the overdose crisis in public libraries across five U.S. states, and the front-line response of public library workers. We conducted a cross-sectional survey, inviting one worker to respond at each public library in five randomly selected states (CO, CT, FL, MI, and VA), querying participants regarding substance use and overdose in their communities and institutions, and their preparedness to respond. We describe substance use and overdose patterns, as well as correlates of naloxone uptake, in public libraries. Participating library staff (N = 356) reported witnessing alcohol use (45%) and injection drug use (14%) in their libraries in the previous month. Across states surveyed, 12% of respondents reported at least one on-site overdose in the prior year, ranging from a low of 10% in MI to a high of 17% in FL. There was wide variation across states in naloxone uptake at libraries, ranging from 0% of represented libraries in FL to 33% in CO. Prior on-site overdose was associated with higher odds of naloxone uptake by the library (OR 2.5, 95% CI 1.1-5.7). Although 24% of respondents had attended a training regarding substance use in the prior year, over 90% of respondents wanted to receive additional training on the topic. Public health professionals should partner with public libraries to expand and strengthen substance use outreach and overdose prevention efforts.

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.

Neoplasia and Proliferative Lesions of New World Camelids: A Systematic Literature Review and Retrospective Study of Cases Submitted to Colorado State University From 1995 to 2020.

Camelid pathology submissions to veterinary diagnostic laboratories are on the rise given the increasing popularity and population of llamas and alpacas especially in the western United States. When compared to other animals, the field of camelid neoplasia has a relative paucity of cases reported in the literature. The Colorado State University Veterinary Diagnostic Laboratories (CSU-VDL) has had a steady increase in the numbers of camelid pathology submissions allowing for a robust review of diagnoses of neoplasia in new world camelids. Here we present a retrospective analysis of camelid neoplastic and proliferative lesions diagnosed at the CSU-VDL from 1995 to 2020, followed by an extensive literature review. Results show increasing incidence of camelid neoplasia reported in the literature, therefore becoming a common diagnosis in llamas and alpacas. Proliferative and neoplastic lesions were diagnosed in 8.8% of new world camelid submissions to CSU-VDL with the most common tumors being lymphomas, squamous cell carcinomas, fibromas, and adenocarcinomas. Risk factors are female sex and increased age except in the case of lymphoma, which tends to occur in younger camelids. Lymphomas, melanomas, and adenocarcinomas (especially of gastrointestinal tract) carry an increased risk of multiple-organ system involvement often with widespread metastases. Conditions described in camelids for the first time include osteosarcoma, cutaneous hemangiosarcoma, myxosarcoma, pilomatricoma, ovarian theca cell tumor, congenital nevus with malignant transformation, and various other neoplasia. This article will provide an operational guide for camelid neoplasia to further assist veterinary laboratory diagnosticians, researchers, and practicing veterinarians in the field of camelid medicine and pathology.

Identification and Characterisation of cis-Regulatory Elements Upstream of the Human Receptor Tyrosine Kinase Gene MERTK.

BACKGROUND: MERTK encodes a receptor tyrosine kinase that regulates immune homeostasis via phagocytosis of apoptotic cells and cytokine-mediated immunosuppression. MERTK is highly expressed in the central nervous system (CNS), specifically in myeloid derived innate immune cells and its dysregulation is implicated in CNS pathologies including the autoimmune disease multiple sclerosis (MS). OBJECTIVE: While the cell types and tissues that express MERTK have been well described, the genetic elements that define the gene's promoter and regulate specific transcription domains remain unknown. The primary objective of this study was to define and characterise the human MERTK promoter region. METHODS: We cloned and characterized the 5' upstream region of MERTK to identify cis-acting DNA elements that promote gene transcription in luciferase reporter assays. In addition, promoter regions were tested for sensitivity to the anti-inflammatory glucocorticoid dexamethasone. RESULTS: This study identified identified both proximal and distal-acting DNA elements that promote transcription. The strongest promoter activity was identified in an ∼850 bp region situated 3 kb upstream of the MERTK transcription start site. Serial deletions of this putative enhancer revealed that the entire region is essential for expression activity. Using in silico analysis, we identified several candidate transcription factor binding sites. Despite a well-established upregulation of MERTK in response to anti-inflammatory glucocorticoids, no DNA region within the 5 kb putative promoter was found to directly respond to dexamethasone treatment. CONCLUSIONS: Elucidating the genetic mechanisms that regulate MERTK expression gives insights into gene regulation during homeostasis and disease, providing potential targets for therapeutic modulation of MERTK transcription.

Corynebacterium mustelae Endocarditis in a Dog.

A 7-year-old male neutered Labrador Retriever dog presented with acute-onset fever, shifting limb lameness and anorexia, with development of acute respiratory distress. At necropsy, there was vegetative endocarditis, which effaced the aortic valve. Gram staining of impression smears from the aortic valve and kidney revealed numerous gram-positive rods with some coryneform bacteria. Similar coryneform bacteria were isolated on aerobic culture of the aortic valve. Identification was attempted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and 16S sequencing, the latter of which indicated Corynebacterium mustelae. This is the second reported case of endocarditis in a dog involving C. mustelae and the first with a description of post-mortem pathology. This case is an example of the utility of various modalities to identify facultative anaerobic bacterial pathogens that may be difficult to culture and may be more widespread than previously diagnosed.

Neural differentiation medium for human pluripotent stem cells to model physiological glucose levels in human brain.

Cortical neurospheres (NSPs) derived from human pluripotent stem cells (hPSC), have proven to be a successful platform to investigate human brain development and neuro-related diseases. Currently, many of the standard hPSC neural differentiation media, use concentrations of glucose (approximately 17.5-25 mM) and insulin (approximately 3.2 µM) that are much greater than the physiological concentrations found in the human brain. These culture conditions make it difficult to analyse perturbations of glucose or insulin on neuronal development and differentiation. We established a new hPSC neural differentiation medium that incorporated physiological brain concentrations of glucose (2.5 mM) and significantly reduced insulin levels (0.86 µM). This medium supported hPSC neural induction and formation of cortical NSPs. The revised hPSC neural differentiation medium, may provide an improved platform to model brain development and to investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels.

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.

Endocrine disrupting chemicals: Impacts on human fertility and fecundity during the peri-conception period.

It is becoming increasingly difficult to avoid exposure to man-made endocrine disrupting chemicals (EDCs) and environmental toxicants. This escalating yet constant exposure is postulated to partially explain the concurrent decline in human fertility that has occurred over the last 50 years. Controversy however remains as to whether associations exist, with conflicting findings commonly reported for all major EDC classes. The primary aim of this extensive work was to identify and review strong peer-reviewed evidence regarding the effects of environmentally-relevant EDC concentrations on adult male and female fertility during the critical periconception period on reproductive hormone concentrations, gamete and embryo characteristics, as well as the time to pregnancy in the general population. Secondly, to ascertain whether individuals or couples diagnosed as sub-fertile exhibit higher EDC or toxicant concentrations. Lastly, to highlight where little or no data exists that prevents strong associations being identified. From the greater than 1480 known EDCs, substantial evidence supports a negative association between exposure to phthalates, PCBs, PBDEs, pyrethroids, organochloride pesticides and male fertility and fecundity. Only moderate evidence exists for a negative association between BPA, PCBs, organochloride pesticides and female fertility and fecundity. Overall fewer studies were reported in women than men, with knowledge gaps generally evident for both sexes for all the major EDC classes, as well as a paucity of female fertility studies following exposure to parabens, triclosans, dioxins, PFAS, organophosphates and pyrethroids. Generally, sub-fertile individuals or couples exhibit higher EDC concentrations, endorsing a positive association between EDC exposure and sub-fertility. This review also discusses confounding and limiting factors that hamper our understanding of EDC exposures on fertility and fecundity. Finally, it highlights future research areas, as well as government, industry and social awareness strategies required to mitigate the negative effects of EDC and environmental toxicant exposure on human fertility and fecundity.