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.

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.

Serum FGF-21, GDF-15, and blood mtDNA copy number are not biomarkers of Parkinson disease.

BACKGROUND: Strong evidence of mitochondrial dysfunction exists for both familial and sporadic Parkinson disease (PD). A simple test, reliably identifying mitochondrial dysfunction, could be important for future stratified medicine trials in PD. We previously undertook a comparison of serum biomarkers in classic mitochondrial diseases and established that serum growth differentiation factor 15 (GDF-15) outperforms fibroblast growth factor 21 (FGF-21) when distinguishing patients with mitochondrial diseases from healthy controls. This study aimed to systematically assess serum FGF-21 and GDF-15, together with mitochondrial DNA (mtDNA) copy number levels in peripheral blood cells from patients with PD and healthy controls, to determine whether these measures could act as a biomarker of PD. METHODS: One hundred twenty-one patients with PD and 103 age-matched healthy controls were recruited from a single center. Serum FGF-21 and GDF-15, along with blood mtDNA copy number, were quantified using established assays. RESULTS: There were no meaningful differences identified for any of the measures when comparing patients with PD with healthy controls. This highlights a lack of diagnostic sensitivity that is incompatible with these measures being used as biomarkers for PD. CONCLUSION: In this study, serum FGF-21, serum GDF-15, and blood mtDNA levels were similar in patients with PD and healthy controls and therefore unlikely to be satisfactory indicators of mitochondrial dysfunction in patients with PD. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum FGF-21, serum GDF-15, and blood mtDNA copy number levels do not distinguish patients with PD from healthy controls. There was no diagnostic uncertainty between patients with PD and healthy controls.

Immunoglobulin G level variations in treated chronic inflammatory demyelinating polyneuropathy: clues for future treatment regimens?

Intravenous immunoglobulins (IVIg) are effective for treating chronic inflammatory demyelinating polyneuropathy (CIDP), although treatment needs are variable and need to be individualized. Dose and frequency requirements are not currently predictable in advance. In Guillain-Barré syndrome, IVIg interpatient pharmacokinetic variations have been demonstrated in relation to clinical outcome. We studied 15 patients with CIDP following two routine courses of IVIg. IgG levels were assessed pretreatment and 14 days post-treatment. Best clinical response (BCR) was ascertained in each case, predefined, according to individual patients' circumstances, on the 10 m walk, or MRC sum score, or Jamar grip dynamometry. Correlations between IgG level variations, doses administered, weight, body mass index, BCR and infusion interval were determined. Postinfusion rise in IgG levels were correlated in individual patients (p = 0.005), but interpatient variability was high. No correlations were ascertained between IgG level variation and weight, body mass index, BCR, total dose of IVIg administered, or dose of IVIg administered per kilogram per week. There were significant correlations between total dose administered and post-infusion IgG level at 14 days (p = 0.004) and between infusion interval and mean rise in IgG level (p = 0.001) These findings suggest significant variability in IgG metabolism between patients, unrelated to minimal effective dose administered, weight, body mass index or degree of functional improvement. Required frequency of IVIg infusions may, however, importantly relate to patient-specific post-infusion rise in IgG levels hence possibly explaining inter-patient differences in treatment frequency needs. IgG level monitoring may be helpful in establishing optimum treatment regimens in individual cases.

Chronic inflammatory pure sensory polyradiculoneuropathy: a rare CIDP variant with unusual electrophysiology.

We describe a patient presenting with progressive upper limb numbness and sensory ataxia of the 4 limbs. Motor nerve conduction studies were completely normal. Sensory electrophysiology showed reduced/absent upper limb sensory action potentials (SAPs). In the lower limbs, SAPs were mostly normal. Sensory conduction velocities were normal. Forearm sensory conduction blocks were present for both median nerves on antidromic testing. The maximal recordable sural SAP was preserved in comparison to maximal recordable radial SAP, consistent with an "abnormal radial normal sural" pattern. Somatosensory evoked potentials were unrecordable for tibial and median nerves. Cerebrospinal fluid protein was raised (0.99 g/L). The patient worsened on oral corticosteroids but subsequently made substantial functional recovery on intravenous immunoglobulins. This case is different to those previously reported of sensory chronic inflammatory demyelinating polyradiculoneuropathy, given its exclusive sensory electrophysiologic presentation, presence of predominant upper limb reduced sensory amplitudes, and detection of sensory conduction blocks. These electrophysiologic features were of paramount importance in establishing diagnosis and effective therapy.

Steroid-induced inflammatory neuropathy in a patient on tumor necrosis factor-α antagonist therapy.

We describe a patient on the tumor necrosis factor-α antagonist, adalimumab, for 2 years for rheumatoid arthitis, who developed a rapidly progressive inflammatory neuropathy shortly after starting oral steroids. Adalimumab was stopped at onset of neurologic symptoms. Electrophysiology showed demyelination, which persisted at 6 month follow-up, cerebrospinal fluid analysis showed persistent albuminocytologic dissociation, and magnetic resonance studies revealed enlarged and enhancing nerve roots. Treatment with intravenous immunoglobulins resulted in slow, progressive improvement. Tumor necrosis factor-α antagonists have previously been implicated in acquired demyelinating neuropathies. In this patient, we hypothesize that adalimumab may have caused an initially asymptomatic chronic inflammatory demyelinating polyneuropathy, which became symptomatic shortly after initiation of steroid therapy. This case may raise the issue of the safety of steroids in conjunction with antitumor necrosis factor-α therapy in susceptible patients.