Protein dose requirements to maximize skeletal muscle protein synthesis after repeated bouts of resistance exercise in young trained women.

Studies examining the effect of protein (PRO) feeding on post resistance exercise (RE) muscle protein synthesis (MPS) have primarily been performed in men, and little evidence is available regarding the quantity of PRO required to maximally stimulate MPS in trained women following repeated bouts of RE. We therefore quantified acute (4 h and 8 h) and extended (24 h) effects of two bouts of resistance exercise, alongside protein-feeding, in women, and the PRO requirement to maximize MPS. Twenty-four RE trained women (26.6 ± 0.7 years, mean ± SEM) performed two bouts of whole-body RE (3 × 8 repetitions/maneuver at 75% 1-repetition maximum) 4 h apart, with post-exercise ingestion of 15 g, 30 g, or 60 g whey PRO (n = 8/group). Saliva, venous blood, and a vastus lateralis muscle biopsy were taken at 0 h, 4 h, 8 h, and 24 h post-exercise. Plasma leucine and branched chain amino acids were quantified using gas chromatography mass spectrometry (GC-MS) after ingestion of D2 O. Fifteen grams PRO did not alter plasma leucine concentration or myofibrillar synthetic rate (MyoFSR). Thirty and sixty grams PRO increased plasma leucine concentration above baseline (105.5 ± 5.3 µM; 120.2 ± 7.4 µM, respectively) at 4 h (151.5 ± 8.2 µM, p < 0.01; 224.8 ± 16.0 µM, p < 0.001, respectively) and 8 h (176.0 ± 7.3 µM, p < 0.001; 281.7 ± 21.6 µM, p < 0.001, respectively). Ingestion of 30 g PRO increased MyoFSR above baseline (0.068 ± 0.005%/h) from 0 to 4 h (0.140 ± 0.021%/h, p < 0.05), 0 to 8 h (0.121 ± 0.012%/h, p < 0.001), and 0 to 24 h (0.099 ± 0.011%/h, p < 0.01). Ingestion of 60 g PRO increased MyoFSR above baseline (0.063 ± 0.003%/h) from 0 to 4 h (0.109 ± 0.011%/h, p < 0.01), 0 to 8 h (0.093 ± 0.008%/h, p < 0.01), and 0 to 24 h (0.086 ± 0.006%/h, p < 0.01). Post-exercise ingestion of 30 g or 60 g PRO, but not 15 g, acutely increased MyoFSR following two consecutive bouts of RE and extended the anabolic window over 24 h. There was no difference between the 30 g and 60 g responses.

Effect of sex and combat employment on musculoskeletal injuries and medical downgrading in trained military personnel: an observational cohort study.

INTRODUCTION: Following the opening of all combat roles to women across the UK Armed Forces, there is a requirement to understand the risk of injury to these female personnel. Women injure at a higher rate than men during basic military training, but fewer data are published from individuals who have passed military training. METHODS: A bespoke survey was designed to investigate differences in injury prevalence and medical downgrading between sexes and career employment groups (ie, job roles) in the UK Armed Forces. RESULTS: Questionnaire data were evaluated from 847 service personnel (87% men) employed in combat roles (Royal Marines, Infantry, Royal Armoured Corps, Royal Air Force Regiment (all men)) and non-combat roles (Royal Regiment of Artillery, Corps of Royal Engineers, Royal Logistic Corps and Combat Service Support Corps who were attached to one of the participating units (men and women)). Women reported more total (OR 1.64 (95% CI: 1.03 to 2.59), p=0.035), lower limb (OR 1.92 (95% CI: 1.23 to 2.98), p=0.004) and hip (OR 2.99 (95% CI: 1.59 to 5.62), p<0.001) musculoskeletal injuries in the previous 12 months than men, but there were no sex differences in the prevalence of current or career medical downgrading due to musculoskeletal injury (both p>0.05). There were no differences in 12-month musculoskeletal injury prevalence between men in combat roles and men in non-combat roles (all p>0.05), but men in non-combat roles were more likely to be currently medically downgraded (OR 1.88 (95% CI: 1.27 to 2.78), p=0.001) and medically downgraded during their career (OR 1.49 (95% CI: 1.11 to 2.00), p=0.008) due to musculoskeletal injury than men in combat roles. More time in service and quicker 1.5-mile run times were associated with increased prevalence of total musculoskeletal injuries, and female sex was a predictor of hip injury. CONCLUSIONS: Although women are at greater risk of injury than men, we have no evidence that combat employment is more injurious than non-combat employment. The prevention of hip injuries should form a specific focus of mitigation efforts for women.

Integrating women into combat roles: comparing the UK Armed Forces and Israeli Defense Forces to understand where lessons can be learnt.

In October 2018, women became eligible to serve in all roles in the UK Armed Forces. The Israeli Defense Forces (IDF) have employed women in limited ground close combat (GCC) roles since 1995. Integration of women into some IDF combat units has improved our understanding of injury risk for servicewomen. While the research findings from physiological studies on women in IDF GCC roles helped inform the integration of women into UK GCC roles, the applicability of the data is limited by the differences between Israeli and UK approaches to recruitment, training and operational deployability of servicewomen. Women in IDF combat roles do, therefore, not provide a good model on the health and performance implications for women in UK Armed Forces combat roles. Further original research to better understand and develop mitigations against any health risks facing UK servicewomen in GCC roles is warranted.

Hormonal contraceptive prescriptions in the UK Armed Forces.

INTRODUCTION: Thirty four per cent of women use hormonal contraceptives in the UK and the contraceptive pill is the most common method. There are no comparable data in the UK Armed Forces, but servicewomen are often required to complete physically arduous job roles in combat zones and may be more likely to take contraceptives to control or stop menstrual bleeding than the general population. We explored the prevalence of hormonal contraceptive prescriptions in the UK Armed Forces. METHODS: The study used defence medical records (Defence Medical Information Capability Programme) to identify hormonal contraceptive prescriptions for all serving regular UK servicewomen (n=15 738) as of 1 September 2017. RESULTS: Thirty one per cent of servicewomen (Royal Navy, 28%; British Army, 30%; Royal Air Force, 34%) had a current prescription for a hormonal contraceptive. Non-officer ranks were more likely to have a prescription for a hormonal contraceptive (32%) than officers (27%) (p<0.01). The contraceptive pill was more commonly prescribed (68%) than long-acting reversible contraceptive methods (32%) (contraceptive injection, 11%; contraceptive implant, 11%; intrauterine device, 10%). CONCLUSION: Prescription data suggest that the prevalence of hormonal contraceptive use in UK servicewomen is comparable with the general UK population. These findings suggest that military service does not influence prevalence or choice of hormonal contraceptives.

Role of sex and stature on the biomechanics of normal and loaded walking: implications for injury risk in the military.

Load carriage and marching 'in-step' are routine military activities associated with lower limb injury risk in service personnel. The fixed pace and stride length of marching typically vary from the preferred walking gait and may result in overstriding. Overstriding increases ground reaction forces and muscle forces. Women are more likely to overstride than men due to their shorter stature. These biomechanical responses to overstriding may be most pronounced when marching close to the preferred walk-to-run transition speed. Load carriage also affects walking gait and increases ground reaction forces, joint moments and the demands on the muscles. Few studies have examined the effects of sex and stature on the biomechanics of marching and load carriage; this evidence is required to inform injury prevention strategies, particularly with the full integration of women in some defence forces. This narrative review explores the effects of sex and stature on the biomechanics of unloaded and loaded marching at a fixed pace and evaluates the implications for injury risk. The knowledge gaps in the literature, and distinct lack of studies on women, are highlighted, and areas that need more research to support evidence-based injury prevention measures, especially for women in arduous military roles, are identified.

Hormonal contraceptive use, bone density and biochemical markers of bone metabolism in British Army recruits.

INTRODUCTION: Hormonal contraceptive use might impair bone health and increase the risk of stress fracture by decreasing endogenous oestrogen production, a central regulator of bone metabolism. This cross-sectional study investigated bone density and biochemical markers of bone metabolism in women taking hormonal contraceptives on entry to basic military training. METHODS: Forty-five female British Army recruits had biochemical markers of bone metabolism, areal bone mineral density (aBMD) and tibial speed of sound (tSOS) measured at the start of basic military training. Participants were compared by their method of hormonal contraception: no hormonal contraception (NONE), combined contraceptive pill (CP) or depot-medroxyprogesterone acetate (DMPA) (20±2.8 years, 1.64±0.63 m, 61.7±6.2 kg). RESULTS: aBMD was not different between groups (p≥0.204), but tSOS was higher in NONE (3%, p=0.014) when compared with DMPA users. Beta C-terminal telopeptide was higher in NONE (45%, p=0.037) and DMPA users (90%, p=0.003) compared with CP users. Procollagen type 1 N-terminal propeptide was higher in DMPA users compared with NONE (43%, p=0.045) and CP users (127%, p=0.001), and higher in NONE compared with CP users (59%, p=0.014). Bone alkaline phosphatase was higher in DMPA users compared with CP users (56%, p=0.044). CONCLUSIONS: DMPA use was associated with increased bone turnover and decreased cortical bone integrity of the tibia. Lower cortical bone integrity in DMPA users was possibly mediated by increased intracortical remodelling, but trabecular bone was not affected by contraceptive use.

Operator and Potential Exposure to Hydrogen Sulfide: A Study of the British Columbia Dairy Industry.

Hydrogen Sulfide (H2S) is produced in manure storage facilities and released during manure agitation. Exposure to this gas presents health risks to workers at daily exposure of greater than 10 parts per million (ppm). Exposure levels on BC dairy farms are unknown. The aim of this study was to document peak H2S exposure levels to operators and bystanders during manure agitation on British Columbia dairy farms to determine if action levels were exceeded. Farms were evaluated at two time points during manure agitation. Both operator exposure and potential exposure sampling were undertaken. Peak hydrogen sulfide levels were measured and categorized as greater than or less than the ceiling level (10 ppm) and action level (5 ppm). Associations between H2S levels above the ceiling level and farm factors were assessed. Measures were recorded at 43 dairy farms with 91 total measures recorded. Action levels were exceeded in 30% of operator measures and 64% of potential exposure measures while 20% of operator and 53% of potential peak H2S measures exceeded 10 ppm. Manure storage facilities were evenly distributed between indoor and outdoor locations with under-barn (43%) and outdoor storage (47%) most common. Sawdust was the most prominent bedding type (65%). Tractor operated propeller was the most common type of agitation equipment (62%). Manure temperature remained a significant predictor in the multivariate model for operator peak exposure. Exposure to H2S above the action level commonly occurs on BC Dairy farms. Given that Worksafe BC requires exposure control plans for H2S levels above 5 ppm, and that on dairy farms, measures of operator and potential H2S exposures were recorded above these levels, best practices to reduce potential H2S exposure are necessary to reduce possible operator and bystander exposure.

Intact olfactory memory in the 5xFAD mouse model of Alzheimer's disease from 3 to 15 months of age.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that causes profound cognitive dysfunction. Deficits in olfactory memory occur in early stages of AD and may be useful in AD diagnosis. The 5xFAD mouse is a commonly used model of AD, as it develops neuropathology, cognitive and sensori-motor dysfunctions similar to those seen in AD. However, olfactory memory dysfunction has not been studied adequately or in detail in 5xFAD mice. Furthermore, despite sex differences in AD prevalence and symptom presentation, few studies using 5xFAD mice have examined sex differences in learning and memory. Therefore, we tested olfactory memory in male and female 5xFAD mice from 3 to 15 months of age using a conditioned odour preference task. Olfactory memory was not impaired in male or female 5xFAD mice at any age tested, nor were there any sex differences. Because early-onset impairments in very long-term (remote) memory have been reported in 5xFAD mice, we trained a group of mice at 3 months of age and tested olfactory memory 90 days later. Very long-term olfactory memory in 5xFAD mice was not impaired, nor was their ability to perform the discrimination task with new odourants. Examination of brains from 5xFAD mice confirmed extensive Aß-plaque deposition spanning the olfactory memory system, including the olfactory bulb, hippocampus, amygdala and piriform cortex. Overall this study indicates that male and female 5xFAD mice do not develop olfactory memory deficits, despite extensive Aß deposition within the olfactory-memory regions of the brain.

Positive adaptation of HPA axis function in women during 44 weeks of infantry-based military training.

BACKGROUND: Basic military training (BMT) is a useful model of prolonged exposure to multiple stressors. 8-12 week BMT is associated with perturbations in the hypothalamic-pituitary-adrenal (HPA) axis which could predispose recruits to injury and psychological strain. However, characterisations of HPA axis adaptations during BMT have not been comprehensive and most studies included few if any women. METHODS: We studied women undertaking an arduous, 44-week BMT programme in the UK. Anxiety, depression and resilience questionnaires, average hair cortisol concentration (HCC), morning and evening saliva cortisol and morning plasma cortisol were assessed at regular intervals throughout. A 1-h dynamic cortisol response to 1 µg adrenocorticotrophic hormone-1-24 was performed during weeks 1 and 29. RESULTS: Fifty-three women (aged 24 ± 2.5 years) completed the study. Questionnaires demonstrated increased depression and reduced resilience during training (F 6.93 and F 7.24, respectively, both p < 0.001). HCC increased from 3 months before training to the final 3 months of training (median (IQR) 9.63 (5.38, 16.26) versus 11.56 (6.2, 22.45) pg/mg, p = 0.003). Morning saliva cortisol increased during the first 7 weeks of training (0.44 ± 0.23 versus 0.59 ± 0.24 µg/dl p < 0.001) and decreased thereafter, with no difference between the first and final weeks (0.44 ± 0.23 versus 0.38 ± 0.21 µg/dl, p = 0.2). Evening saliva cortisol did not change. Fasting cortisol decreased during training (beginning, mid and end-training concentrations: 701 ± 134, 671 ± 158 and 561 ± 177 nmol/l, respectively, p < 0.001). Afternoon basal cortisol increased during training while there was a trend towards increased peak stimulated cortisol (177 ± 92 versus 259 ± 13 nmol/l, p = 0.003, and 589 ± 164 versus 656 ± 135, p = 0.058, respectively). DISCUSSION: These results suggest a normal stress response in early training was followed quickly by habituation, despite psychological and physical stress evidenced by questionnaire scores and HCC, respectively. There was no evidence of HPA axis maladaptation. These observations are reassuring for women undertaking arduous employment.

Cardiorespiratory coordination reveals training-specific physiological adaptations.

PURPOSE: To compare the effects of high-intensity interval training (HIIT) and moderate-intensity training (CONT), matched for total work, on cardiorespiratory coordination and aerobic fitness. METHODS: This is a two-arm parallel group single-blind randomised study. Twenty adults were assigned to 6 weeks of HIIT or volume-matched CONT. Participants completed a progressive maximal cycling test before and after the training period. Principal component (PC) analysis was performed on the series of cardiorespiratory variables to evaluate dimensionality of cardiorespiratory coordination, before and after lactate turnpoint. PC1 eigenvalues were compared. RESULTS: Both HIIT and CONT improved aerobic fitness (main effects of time, p < 0.001, [Formula: see text] ≥ 0.580), with no differences between groups. CONT decreased the number of PCs from two to one at intensities both below and above the lactate turnpoint; PC1 eigenvalues increased after CONT both below (Z = 2.08; p = 0.04; d = 0.94) and above the lactate turnpoint (Z = 2.10; p = 0.04; d = 1.37). HIIT decreased the number of PCs from two to one after the lactate turnpoint only; PC1 eigenvalues increased after HIIT above the lactate turnpoint (Z = 2.31; p = 0.02; d = 0.42). CONCLUSIONS: Although CONT and HIIT improved aerobic fitness to a similar extent, there were different patterns of change for cardiorespiratory coordination. These changes appear training-intensity specific and could be sensitive to investigate the individual response to endurance training.

Motor function deficits in the 12 month-old female 5xFAD mouse model of Alzheimer's disease.

Motor problems occur early in some patients with Alzheimer's disease (AD) and as the disease progresses many patients develop motor dysfunction. Motor dysfunction has been reported in some mouse models of AD, including the 5xFAD mouse, thus this model may be particularly useful for studying motor dysfunction in AD. In order to determine the extent of motor dysfunction in these mice, we tested 11-13 month old female 5xFAD and wildtype (WT) control mice in a battery of motor behaviour tasks. The 5xFAD mice showed hind limb clasping, weighed less and had slower righting reflexes than WT mice. In the open field, the 5xFAD mice travelled a shorter distance than the WT mice, spent less time moving and had a slower movement speed. The 5xFAD mice fell faster than the WT mice from the balance beam, wire suspension, grid suspension and rotarod tasks, indicating dysfunctions in balance, grip strength, motor co-ordination and motor learning. The 5xFAD mice had a short, shuffling gait with a shorter stride length than WT mice and had a slower swim speed. The 5xFAD mice also failed to show an acoustic startle response, likely due to motor dysfunction and previously reported hearing impairment. The 5xFAD mice did not show deficits in the ability of peripheral motor nerves to drive muscle output, suggesting that motor impairments are not due to dysfunction in peripheral motor nerves. These results indicate that the aged 5xFAD mice are deficient in numerous motor behaviours, and suggest that these mice may prove to be a good model for studying the mechanisms of motor dysfunction in AD, and motor behaviour might prove useful for assessing the efficacy of AD therapeutics. Motor dysfunction in 5xFAD mice must also be considered in behavioural tests of sensory and cognitive function so that performance is not confounded by impaired locomotor or swimming behaviour.

Endurance capacity and neuromuscular fatigue following high- vs moderate-intensity endurance training: A randomized trial.

High-intensity exercise induces significant central and peripheral fatigue; however, the effect of endurance training on these mechanisms of fatigue is poorly understood. We compared the effect of cycling endurance training of disparate intensities on high-intensity exercise endurance capacity and the associated limiting central and peripheral fatigue mechanisms. Twenty adults were randomly assigned to 6 weeks of either high-intensity interval training (HIIT, 6-8×5 minutes at halfway between lactate threshold and maximal oxygen uptake [50%Δ]) or volume-matched moderate-intensity continuous training (CONT, ~60-80 minutes at 90% lactate threshold). Two time to exhaustion (TTE) trials at 50%Δ were completed pre- and post-training to assess endurance capacity; the two post-training trials were completed at the pretraining 50%Δ (same absolute intensity) and the "new" post-training 50%Δ (same relative intensity). Pre- and post-exercise responses to femoral nerve and motor cortex stimulation were examined to determine peripheral and central fatigue, respectively. HIIT resulted in greater increases in TTE at the same absolute and relative intensities as pre-training (148% and 43%, respectively) compared with CONT (38% and -4%, respectively) (P≤.019). Compared with pre-training, HIIT increased the level of potentiated quadriceps twitch reduction (-34% vs -43%, respectively, P=.023) and attenuated the level of voluntary activation reduction (-7% vs -3%, respectively, P=.047) following the TTE trial at the same relative intensity. There were no other training effects on neuromuscular fatigue development. This suggests that central fatigue resistance contributes to enhanced high-intensity exercise endurance capacity after HIIT by allowing greater performance to be extruded from the muscle.

Reduced acoustic startle response and peripheral hearing loss in the 5xFAD mouse model of Alzheimer's disease.

Hearing dysfunction has been associated with Alzheimer's disease (AD) in humans, but there is little data on the auditory function of mouse models of AD. Furthermore, characterization of hearing ability in mouse models is needed to ensure that tests of cognition that use auditory stimuli are not confounded by hearing dysfunction. Therefore, we assessed acoustic startle response and pre-pulse inhibition in the double transgenic 5xFAD mouse model of AD from 3-4 to 16 months of age. The 5xFAD mice showed an age-related decline in acoustic startle as early as 3-4 months of age. We subsequently tested auditory brainstem response (ABR) thresholds at 4 and 13-14 months of age using tone bursts at frequencies of 2-32 kHz. The 5xFAD mice showed increased ABR thresholds for tone bursts between 8 and 32 kHz at 13-14 months of age. Finally, cochleae were extracted and basilar membranes were dissected to count hair cell loss across the cochlea. The 5xFAD mice showed significantly greater loss of both inner and outer hair cells at the apical and basal ends of the basilar membrane than wild-type mice at 15-16 months of age. These results indicate that the 5xFAD mouse model of AD shows age-related decreases in acoustic startle responses, which are at least partially due to age-related peripheral hearing loss. Therefore, we caution against the use of cognitive tests that rely on audition in 5xFAD mice over 3-4 months of age, without first confirming that performance is not confounded by hearing dysfunction.

Central and peripheral fatigue following non-exhaustive and exhaustive exercise of disparate metabolic demands.

The development of fatigue after non-exhaustive and exhaustive exercise eliciting differing metabolic demands is poorly understood. Sixteen active males completed five cycling trials. The first trial established the lactate threshold (LT) and maximal oxygen uptake (VO2max ). Two of the remaining trials were completed at a severe intensity (halfway between LT and VO2max , SI) and two at a moderate intensity (90% LT, MI). Each trial involved two non-exhaustive bouts matched for work between intensities before cycling to exhaustion. Responses to stimulation of the femoral nerve and motor cortex were determined after each bout to determine peripheral and central fatigue. Corticospinal excitability, cortical silent period (cSP), short-interval intracortical inhibition (SICI), and intracortical facilitation (ICF) were also assessed. Non-exhaustive cycling induced greater peripheral and central fatigue in the SI compared with the MI (P < 0.05). At exhaustion, there was no difference between intensities; however, peripheral fatigue tended to be greater in the SI vs MI (-31% vs -17%, respectively, P = 0.051). Exhaustive cycling increased SICI (24%, P < 0.001) and reduced the cSP (-14%, P < 0.001) in the SI, whereas ICF was reduced in the MI (-16%, P < 0.001). These findings demonstrate exercise-induced metabolic stress accelerates the development of peripheral and central fatigue, and differentially influences intracortical excitability.

Early detection of cerebral glucose uptake changes in the 5XFAD mouse.

Brain glucose hypometabolism has been observed in Alzheimer's disease (AD) patients, and is detected with (18)F radiolabelled glucose, using positron emission tomography. A pathological hallmark of AD is deposition of brain ß- amyloid plaques that may influence cerebral glucose metabolism. The five times familial AD (5XFAD) mouse is a model of brain amyloidosis exhibiting AD-like phenotypes. This study examines brain ß-amyloid plaque deposition and (18)FDG uptake, to search for an early biomarker distinguishing 5XFAD from wild-type mice. Thus, brain (18)FDG uptake and plaque deposition was studied in these mice at age 2, 5 and 13 months. The 5XFAD mice demonstrated significantly reduced brain (18)FDG uptake at 13 months relative to wild-type controls but not in younger mice, despite substantial ß- amyloid plaque deposition. However, by comparing the ratio of uptake values for glucose in different regions in the same brain, 5XFAD mice could be distinguished from controls at age 2 months. This method of measuring altered glucose metabolism may represent an early biomarker for the progression of amyloid deposition in the brain. We conclude that brain (18)FDG uptake can be a sensitive biomarker for early detection of abnormal metabolism in the 5XFAD mouse when alternative relative uptake values are utilized.

Spontaneous Complete Uterine Rupture in a Nonlaboring, Early Third-trimester Uterus: Missed Diagnosis by Ultrasound.

Objective Describe potential consequences and remedy for not intermittently auscultating fetal heart tones during travel to the imaging department and misdiagnosing a ruptured uterus on ultrasound as a synechiae. Study Design Retrospective chart review of case. Results Spontaneous uterine rupture in pregnancy is a rare and catastrophic event. Fetal monitoring is an important component for diagnosis, but fetal heart auscultation is usually discontinued while the patient is receiving imaging. We present a ruptured uterus at 28 weeks with delayed diagnosis secondary to interrupted fetal heart tone auscultation and a misdiagnosis of a rupture as a synechiae resulting in a seriously compromised newborn at delivery. Conclusion Intermittent fetal heart tone auscultation is a possible method of monitoring when the patient is off continuous fetal monitoring.

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

Effect of small molecule supplements during in vitro culture of mouse zygotes and parthenogenetic embryos on hypoblast formation and stem cell derivation.

BACKGROUND: Small molecule inhibitors are organic components that modulate signalling pathways and have the ability to change the differentiation state of cells. They have been used to increase the efficiency of induced pluripotent stem cell generation and to support stem cell derivation and culture. In this study, we aimed to evaluate the effects of small molecules on the development of mouse zygotes and parthenogenetic embryos. METHODS AND RESULTS: Three inhibitors (SC-1, PD0325901 and BIO) were added to the culture medium from the 2-cell stage onwards. We have observed that addition of an inhibitor of the fibroblast growth factor (FGF) pathway (SC-1 or PD0325901) compromises the segregation of hypoblast from the inner cell mass (ICM). Given no difference was observed in size of the ICM, but more epiblast cells were found in these embryos, we can conclude that this is caused by redirection of all ICM cells to the epiblast. We also determined the consequences of reduced hypoblast and increased epiblast formation on stem cell derivation efficiency. No significant difference was found between derivation rates from treated embryos as compared to controls. However, only under 2i + ROCKi conditions, stem cells could be derived with an efficiency of more than 90%. Addition of BIO, an activator of the WNT pathway, did not have any effects on hypoblast development or stem cell derivation. CONCLUSION: We have demonstrated that FGF signalling is crucial for hypoblast generation and small molecules can be efficiently used to inhibit this process both in zygotes and parthenogenetic embryos.

The influence of patient and cohort parameters on the incidence and developmental potential of embryos with poor quality traits for use in human embryonic stem cell derivation.

BACKGROUND: Human embryonic stem cells (hESCs) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos. While the majority of hESC lines originate from good-quality embryos donated after cryogenic storage, poor-quality embryos (PQEs) not suitable for clinical use have also been shown to generate hESC. This provides a newfound function for embryos that would otherwise be discarded following IVF or ICSI. Owing to their lack of clinical importance, however, data on the poorest embryos in a cohort go largely unreported in the literature. It is therefore of interest to better understand the availability of PQEs from IVF/ICSI cycles and to determine their ability to develop into blastocysts with good-quality ICMs for use in hESC derivation. In this study, we investigate the influence of patient parameters and embryo cohort on PQE incidence, blastocyst development, ICM quality and successful hESC derivation from donated PQEs. METHODS: PQEs from 736 patient cycles that did not meet our clinical criteria for transfer or cryopreservation were cultured until Day 6 of development and assessed for blastocyst formation and ICM quality. A subset of blastocysts with good-quality ICMs were then used for hESC derivation attempts. Anonymous patient data such as maternal age, embryo history and cohort parameters were then retrospectively compiled and analysed. RESULTS: PQEs made up 46.8% of two pronucleate embryos created from IVF/ICSI. Including embryos with abnormal fertilization, a mean of 3.6 ± 2.8 embryos were donated per cycle with 32.6% developing to the blastocyst stage. Good-quality ICM were produced in 13.9% of PQEs cultured. Of good-quality ICM, 15.4% of those used in hESC derivation attempts resulted in a novel line. The PQEs that originated from older patients (>37 year) or from cycles that did not result in pregnancy had significantly diminished blastocyst development and ICM quality. Maternal age was also shown to further influence the ability of good-quality ICMs to generate hESC. CONCLUSIONS: PQEs are an abundant source of embryos capable of developing to blastocysts with good-quality ICMs and subsequently generating novel hESC. We have shown that prognostic variables used to predict IVF/ICSI outcome can also help predict which PQEs have the best hESC developmental potential. Owing to the diversity of PQE origin, experiments designed to compare hESC derivation techniques or efficiency using PQEs should consider clinical IVF/ICSI parameters to establish groups with equal developmental competence. Additional investigation is needed to determine if these results are applicable to hESC derivation using good-quality embryos.