Differential impacts of gonadotrophins, IVF and embryo culture on mouse blastocyst development.

RESEARCH QUESTION: Conception via assisted reproductive technology (ART) increases the risk of type 2 diabetes and cardiovascular disease in adulthood. Underlying differences between ART-conceived and in-vivo-conceived embryos that contribute to this increased risk are, however, not known. DESIGN: This study examined the developmental characteristics of mouse blastocysts derived from ART- compared with in-vivo-conceived embryos. To determine the effect of ovarian stimulation versus IVF versus in-vitro embryo culture on phenotype, six distinct groups of blastocysts were generated. Female mice were naturally cycling or treated with high or mild doses of gonadotrophin, followed by natural mating or IVF under clinical conditions. Embryo morphokinetics were assessed by continuous time-lapse monitoring. Cell lineage allocation to the inner cell mass (Oct4+) or trophectoderm (Cdx2+) was determined by immunohistochemistry, and mitochondrial DNA (mtDNA) copy number was measured by quantitative PCR. RESULTS: Ovarian stimulation increased embryo number but reduced the percentage of blastocysts. Morphokinetic analysis showed that gonadotrophin treatment led to advanced development (P < 0.05) due to earlier post-pronuclear breakdown. The blastocyst rate was reduced in IVF embryos compared with those fertilized in vivo before culture (P < 0.001). Morphokinetics showed that embryo development was slower in all the IVF groups (P < <0.05), due to a delay from the 3-cell stage. A reduced total and trophectoderm cell number was observed in all groups of cultured blastocysts compared with naturally conceived blastocysts (P < 0.01). Gonadotrophin treatment did not affect the blastocyst mtDNA copy number; however, IVF embryos exhibited reduced mtDNA copy number compared with naturally conceived embryos. CONCLUSION: Ovarian stimulation, IVF and in-vitro culture differentially impair blastocyst developmental kinetics, differentiation and mtDNA copy number.

Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

Over-nutrition in females causes altered fetal growth during pregnancy and permanently programs the metabolism of offspring; however, the temporal and mechanistic origins of these changes, and whether they are reversible, are unknown. We now show that, in obese female mice, cumulus-oocyte complexes exhibit endoplasmic reticulum (ER) stress, high levels of intracellular lipid, spindle abnormalities and reduced PTX3 extracellular matrix protein production. Ovulated oocytes from obese mice contain normal levels of mitochondrial (mt) DNA but have reduced mitochondrial membrane potential and high levels of autophagy compared with oocytes from lean mice. After in vitro fertilization, the oocytes of obese female mice demonstrate reduced developmental potential and form blastocysts with reduced levels of mtDNA. Blastocysts transferred to normal weight surrogates that were then analyzed at E14.5 showed that oocytes from obese mice gave rise to fetuses that were heavier than controls and had reduced liver and kidney mtDNA content per cell, indicating that maternal obesity before conception had altered the transmission of mitochondria to offspring. Treatment of the obese females with the ER stress inhibitor salubrinal or the chaperone inducer BGP-15 before ovulation increased the amount of the mitochondrial replication factors TFAM and DRP1, and mtDNA content in oocytes. Salubrinal and BGP-15 also completely restored oocyte quality, embryo development and the mtDNA content of fetal tissue to levels equivalent to those derived from lean mice. These results demonstrate that obesity before conception imparts a legacy of mitochondrial loss in offspring that is caused by ER stress and is reversible during the final stages of oocyte development and maturation.

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 µM), oleic acid (200 µM), and steric acid (75 µM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Hyperglycaemia and lipid differentially impair mouse oocyte developmental competence.

Maternal diabetes and obesity are characterised by elevated blood glucose, insulin and lipids, resulting in upregulation of specific fuel-sensing and stress signalling pathways. Previously, we demonstrated that, separately, upregulation of the hexosamine biosynthetic pathway (HBP; under hyperglycaemic conditions) and endoplasmic reticulum (ER) stress (due to hyperlipidaemia) pathways reduce blastocyst development and alter oocyte metabolism. In order to begin to understand how both glucose and lipid metabolic disruptions influence oocyte developmental competence, in the present study we exposed mouse cumulus-oocyte complexes to hyperglycaemia (30mM) and/or lipid (40µM) and examined the effects on embryo development. The presence of glucosamine (GlcN; a hyperglycaemic mimetic) or increased lipid during in vitro maturation severely perturbed blastocyst development (P<0.05). Hyperglycaemia, GlcN and hyperglycaemia + lipid treatments significantly increased HBP activity, increasing total O-linked glycosylation (O-GlcNAcylation) of proteins (P<0.0001). All treatments also induced ER stress pathways, indicated by the expression of specific ER stress genes. The expression of genes encoding the HBP enzymes glutamine:fructose-6-phosphate amidotransferase 2 (Gfpt2) and O-linked ß-N-acetylglucosaminyltransferase (Ogt) was repressed following lipid treatment (P<0.001). These findings partially implicate the mechanism of O-GlcNAcylation and ER stress as likely contributors to compromised fertility of obese women.

Distinct localisation of lipids in the ovarian follicular environment.

Obesity is associated with decreased pregnancy rates due, in part, to compromised oocyte quality. The aim of the present cross-sectional study of 84 women undergoing oocyte aspiration was to: (1) compare insulin, lipids and glucose in follicular fluid with serum; (2) determine whether increased body mass index (BMI) and waist circumference, hyperinsulinaemia, dyslipidaemia or metabolic syndrome altered follicular fluid metabolites; and (3) determine relative lipid content in oocytes to reveal any influence of these parameters on oocyte quality and IVF outcomes. Insulin, glucose, triglyceride and free fatty acids were lower in follicular fluid than blood and not strictly correlated between compartments. Insulin, glucose and triglyceride positively correlated with increasing BMI and waist circumference in blood and follicular fluid. Insulin increased in follicular fluid in association with metabolic syndrome. Free fatty acid composition analysis showed saturated fatty acids, particularly palmitic and stearic acid, to be more prevalent in follicular fluid than blood. There were no associations between follicular fluid metabolites or oocyte lipid content and clinical outcomes; however, oocyte immaturity correlated with follicular fluid glucose and fatty acid levels, as well as metabolic syndrome. The present study confirms that the human ovarian follicular environment surrounding the oocyte exhibits a unique metabolite profile compared with blood, with distinct localisation of lipids within follicular fluid and oocytes.

Female offspring sired by diet induced obese male mice display impaired blastocyst development with molecular alterations to their ovaries, oocytes and cumulus cells.

PURPOSE: To investigate the impacts that a paternal high fat diet (HFD) has on embryology, ovarian/cumulus cell gene expression and COC metabolism from female offspring, using a mouse model. METHODS: Founder male mice were either fed a control diet (CD) or a HFD for 12 weeks. The HFD induced obesity but not diabetes, and founder males were then mated to normal weight CD fed female mice. Female offspring were maintained on a CD, super-ovulated, mated and the resultant zygotes were cultured to the blastocyst stage for embryo morphology, blastocyst cell number and apoptosis assessment. Ovaries and cumulus cells from offspring were collected for gene expression analysis of selected genes that maintain chromatin remodeling and endoplasmic reticulum (ER), metabolic and inflammatory homeostasis. Cumulus/oocyte complexes were also investigated for glucose uptake and lipid accumulation. RESULTS: Female offspring sired by obese fathers produced embryos with delayed development and impaired quality, displayed increases in ovarian expression of Glut1, Glut3 and Glut4, and an increase in cumulus cell expression of Glut4. Interestingly their COCs did take up more glucose, but did accumulate more lipid. CONCLUSIONS: A paternal HFD is associated with subfertility in female offspring despite the offspring being fed a CD and this subfertility is concomitant with ovarian/cumulus cell molecular alterations and increased lipid accumulation.

Clinician Perspectives on the Impact of COVID-19 on the Treatment of Adults and Youth with Anxiety.

We describe the perceptions of mental health clinicians practicing in the United States about the effects of the COVID-19 pandemic on the presentation and treatment course of active clients with anxiety. Clinician participants reported on client symptomology at the beginning of treatment, just before (prior to March 2020), and at a mid-pandemic timepoint (December 2020/January 2021). An initial sample of 70 clinicians responded to a survey assessing their clients' overall anxiety severity, anxiety sensitivity, pathological uncertainty, family accommodation, and avoidance levels. Of these, 54 clinician responses were included in study analyses, providing detailed clinical information on 81 clients. Findings suggest that the COVID-19 pandemic was associated with increases in anxiety severity in the majority of clients; overall, clinicians reported that 53% of clients had symptoms worsen due to COVID-19 and that only 16% experienced improvement of symptoms during treatment. Those who had lower levels of avoidance pre-pandemic and those who increased their frequency of treatment were more likely to experience increases in anxiety severity by the mid-pandemic timepoint. Further research is needed to understand the extended effects of the COVID-19 pandemic on anxiety symptomology and treatment.

The impact of obesity on oocytes: evidence for lipotoxicity mechanisms.

Obesity can have detrimental effects on pregnancy rates in natural conceptions and also in women undergoing IVF or intracytoplasmic sperm injection (ICSI). This review summarises the most recent clinical literature investigating whether obesity impacts oocyte quality and early embryo growth. In other tissues, obesity leads to lipotoxicity responses including endoplasmic reticulum stress, mitochondrial dysfunction and apoptosis. Recent reports indicate that lipotoxicity is a mechanism by which obesity may impact oocyte quality.

Identification of perilipin-2 as a lipid droplet protein regulated in oocytes during maturation.

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus-oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins perilipin, perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only perilipin-2 was associated with lipid droplets in the oocyte. In COCs, perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

Endoplasmic reticulum (ER) stress in cumulus-oocyte complexes impairs pentraxin-3 secretion, mitochondrial membrane potential (DeltaPsi m), and embryo development.

Fatty acids such as palmitic acid at high levels are known to induce endoplasmic reticulum (ER) stress and lipotoxicity in numerous cell types and thereby contribute to cellular dysfunctions in obesity. To understand the impact of high fatty acids on oocytes, ER stress and lipotoxicity were induced in mouse cumulus-oocyte complexes during in vitro maturation using the ER Ca(2+) channel blocker thapsigargin or high physiological levels of palmitic acid; both of which significantly induced ER stress marker genes (Atf4, Atf6, Xbp1s, and Hspa5) and inositol-requiring protein-1α phosphorylation, demonstrating an ER stress response that was reversible with the ER stress inhibitor salubrinal. Assessment of pentraxin-3, an extracellular matrix protein essential for fertilization, by immunocytochemistry and Western blotting showed dramatically impaired secretion concurrent with ER stress. Mitochondrial activity in oocytes was assessed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining of inner mitochondrial membrane potential, and oocytes matured in thapsigargin or high-dose palmitic acid had significantly reduced mitochondrial activity, reduced in vitro fertilization rates, and were slower to develop to blastocysts. The deficiencies in protein secretion, mitochondrial activity, and oocyte developmental competence were each normalized by salubrinal, demonstrating that ER stress is a key mechanism mediating fatty acid-induced defects in oocyte developmental potential.

Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes.

OBJECTIVE: To determine whether the high lipid content of human follicular fluid influences oocyte maturation. DESIGN: Mouse oocytes as substitutes for human oocytes were exposed to follicular fluids of differing lipid content with outcome monitoring. SETTING: Private infertility clinic and university laboratory. PATIENT(S): Seventy-four women seeking assisted reproduction, and gonadotropin-stimulated mice. INTERVENTION(S): Assay of follicular fluids for triglyceride and free fatty acids, and stimulation of mouse cumulus-oocyte complexes (COCs) to maturity in vitro in the presence of lipid-rich or lipid-poor follicular fluid. MAIN OUTCOME MEASURE(S): Oocyte lipid content, expression of endoplasmic reticulum stress marker genes, and oocyte maturation assessed in mouse COCs exposed to lipid-rich follicular fluid were compared with complexes exposed to lipid-poor follicular fluid and complexes matured in vivo. RESULT(S): Follicular fluids were obtained from women of known body mass index undergoing oocyte aspiration at a private infertility clinic, and the follicular fluids were assayed for triglyceride and free fatty acids; those with the highest and lowest levels of these lipids were selected. The mouse COCs exposed to lipid-rich follicular fluid during their maturation had increased oocyte lipid content, induction of endoplasmic reticulum stress markers, and impaired oocyte nuclear maturation. CONCLUSION(S): Increased body mass index is associated with elevated triglycerides and free fatty acids in ovarian follicular fluid. Maturation within this lipid-rich environment is detrimental to oocytes.

Inflammatory pathways linking obesity and ovarian dysfunction.

This review summarizes some of the recent advances in obesity research and describes how we and others have built upon these findings to better understand the impact of obesity on granulosa cells, cumulus cells and oocytes within the ovaries of obese females. Obesity is associated with lipid accumulation in non-adipose tissue cells and the induction of oxidative stress and endoplasmic reticulum stress responses that are tightly linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the ovary in response to obesity. Studies in mice support this and allow further dissection of the pathways by which diet-induced obesity contributes to changes in mitochondria and the endoplasmic reticulum. These studies are in their infancy but cumulatively provide basic information about the cellular mechanisms that may lead to the impaired ovulation and reduced oocyte developmental potential that is observed in obese females.

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice.

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.