Examining well-being and cognitive function in people with long Covid and ME/CFS, and age-matched healthy controls: A Case-Case-Control Study.

PURPOSE: Well-being and cognitive function had not previously been compared between people with long COVID and people with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Therefore, this study examined well-being and cognitive function in people with long COVID (∼16 months illness duration; n= 17) and ME/CFS (∼16 years illness duration; n=24), versus age-matched healthy controls (n=16). METHODS: Well-being was examined using several questionnaires, namely the Health Visual Analogue Scale (VAS), Fatigue Severity Scale (FSS), Post-exertional malaise (PEM), Pittsburgh Sleep Quality Index (PSQI), European Quality of Life-5 Domains (EQ-5D), MRC Dyspnoea, Self-Efficacy (SELTC), The Edinburgh Neurosymptoms Questionnaire (ENS), General Anxiety Disorder 7 (GAD-7), and Patient Health Questionnaire 9 (PHQ-9). Cognitive function was examined using Single Digit Modalities Test (SDMT), Stroop test, and Trails A and B. These were delivered via a mobile application (app) built specifically for this remote data collection. RESULTS: The main findings of the present investigation were that people with ME/CFS and people with long COVID were generally comparable on all well-being and cognitive function measures, but self-reported worse values for pain, fatigue, Post-exertional malaise, sleep quality, general well-being in relation to mobility, usual activities, self-care, breathlessness, neurological symptoms, self-efficacy, and other well-being such as anxiety and depression, compared to controls. There was no effect of group for cognitive function measures. CONCLUSIONS: These data suggest that both people with long COVID and people with ME/CFS have similar impairment on well-being measures examined herein. Therefore, interventions that target well-being of people with ME/CFS and long COVID are required.

People with Long COVID and ME/CFS Exhibit Similarly Impaired Dexterity and Bimanual Coordination: A Case-Case-Control Study.

PURPOSE: Dexterity and bimanual coordination had not previously been compared between people with long COVID and people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Therefore, this study determined dexterity and bimanual coordination in people with long COVID (∼16-month illness duration; n = 21) and ME/CFS (∼16-year illness duration; n = 20), vs age-matched healthy controls (n = 20). METHODS: Dexterity and bimanual coordination was determined using the Purdue pegboard test. RESULTS: The main findings of the present investigation were that people with ME/CFS and people with long COVID were generally comparable for Purdue pegboard tests (P > .556 and d < 0.36 for pairwise comparisons). It is worth noting however, that both these patient groups performed poorer in the Perdue pegboard test than healthy controls (P < .169 and d > 0.40 for pairwise comparisons). CONCLUSIONS: These data suggest that both people with long COVID and people with ME/CFS have similarly impaired dexterity and bimanual coordination. Therefore, there is an urgent need for interventions to target dexterity and bimanual coordination in people with ME/CFS, and given the current pandemic, people with long COVID.

Post-Traumatic Stress Disorder and Complex Post-Traumatic Stress Disorder in People with Long COVID, ME/CFS, and Controls.

BACKGROUND: Prevalences of post-traumatic stress disorder (PTSD) and complex post-traumatic stress disorder (CPTSD) have not previously been compared between individuals with long coronavirus disease (COVID) and individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and healthy age-matched controls. For these reasons, this study aimed to determine the prevalence of PTSD and CPTSD in individuals with long COVID (n = 21) and ME/CFS (n = 20) and age-matched controls (n = 20). METHODS: A case-case-control approach was employed; participants completed the International Trauma Questionnaire, a self-report measure of the International Classification of Diseases of PTSD and CPTSD consisting of 18 items. Scores were calculated for each PTSD and Disturbances in Self-Organization (DSO) symptom cluster and summed to produce PTSD and DSO scores. PTSD was diagnosed if the criteria for PTSD were met but not DSO, and CPTSD was diagnosed if the criteria for PTSD and DSO were met. Moreover, each cluster of PTSD and DSO were compared among individuals with long COVID, ME/CFS, and healthy controls. RESULTS: Individuals with long COVID (PTSD = 5%, CPTSD = 33%) had more prevalence of PTSD and CPTSD than individuals with ME/CFS (PTSD = 0%, CPTSD = 20%) and healthy controls (PTSD = 0%, CPTSD = 0%). PTSD and CPTSD prevalence was greater in individuals with long COVID and ME/CFS than controls. Individuals with long COVID had greater values controls for all PTSD values. Moreover, individuals with long COVID had greater values than controls for all DSO values. Individuals with ME/CFS had greater values than controls for all DSO values. Both long COVID and ME/CFS groups differed in overall symptom scores compared with controls. CONCLUSION: Findings of this study demonstrated that individuals with long COVID generally had more cases of PTSD and CPTSD than individuals with ME/CFS and healthy controls.

People with Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Exhibit Similarly Impaired Vascular Function.

BACKGROUND: This study aimed to compare flow-mediated dilation values between individuals with long COVID, individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and healthy age-matched controls to assess the potential implications for clinical management and long-term health outcomes. METHODS: A case-case-control approach was employed, and flow-mediated dilation measurements were obtained from 51 participants (17 long COVID patients, 17 ME/CFS patients, and 17 healthy age-matched controls). Flow-mediated dilation values were analyzed using 1-way analysis of variance for between-group comparisons. RESULTS: Results revealed significantly impaired endothelial function in both long COVID and ME/CFS groups compared with healthy age-matched controls as determined by maximum % brachial artery diameter post-occlusion compared with pre-occlusion resting diameter (6.99 ± 4.33% and 6.60 ± 3.48% vs 11.30 ± 4.44%, respectively, both P < .05). Notably, there was no difference in flow-mediated dilation between long COVID and ME/CFS groups (P = .949), despite significantly longer illness duration in the ME/CFS group (ME/CFS: 16 ± 11.15 years vs long COVID: 1.36 ± 0.51 years, P < .0001). CONCLUSION: The study demonstrates that both long COVID and ME/CFS patients exhibit similarly impaired endothelial function, indicating potential vascular involvement in the pathogenesis of these post-viral illnesses. The significant reduction in flow-mediated dilation values suggests an increased cardiovascular risk in these populations, warranting careful monitoring and the development of targeted interventions to improve endothelial function and mitigate long-term health implications.

A scoping review of 'Pacing' for management of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): lessons learned for the long COVID pandemic.

BACKGROUND: Controversy over treatment for people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a barrier to appropriate treatment. Energy management or pacing is a prominent coping strategy for people with ME/CFS. Whilst a definitive definition of pacing is not unanimous within the literature or healthcare providers, it typically comprises regulating activity to avoid post exertional malaise (PEM), the worsening of symptoms after an activity. Until now, characteristics of pacing, and the effects on patients' symptoms had not been systematically reviewed. This is problematic as the most common approach to pacing, pacing prescription, and the pooled efficacy of pacing was unknown. Collating evidence may help advise those suffering with similar symptoms, including long COVID, as practitioners would be better informed on methodological approaches to adopt, pacing implementation, and expected outcomes. OBJECTIVES: In this scoping review of the literature, we aggregated type of, and outcomes of, pacing in people with ME/CFS. ELIGIBILITY CRITERIA: Original investigations concerning pacing were considered in participants with ME/CFS. SOURCES OF EVIDENCE: Six electronic databases (PubMed, Scholar, ScienceDirect, Scopus, Web of Science and the Cochrane Central Register of Controlled Trials [CENTRAL]) were searched; and websites MEPedia, Action for ME, and ME Action were also searched for grey literature, to fully capture patient surveys not published in academic journals. METHODS: A scoping review was conducted. Review selection and characterisation was performed by two independent reviewers using pretested forms. RESULTS: Authors reviewed 177 titles and abstracts, resulting in 17 included studies: three randomised control trials (RCTs); one uncontrolled trial; one interventional case series; one retrospective observational study; two prospective observational studies; four cross-sectional observational studies; and five cross-sectional analytical studies. Studies included variable designs, durations, and outcome measures. In terms of pacing administration, studies used educational sessions and diaries for activity monitoring. Eleven studies reported benefits of pacing, four studies reported no effect, and two studies reported a detrimental effect in comparison to the control group. CONCLUSIONS: Highly variable study designs and outcome measures, allied to poor to fair methodological quality resulted in heterogenous findings and highlights the requirement for more research examining pacing. Looking to the long COVID pandemic, our results suggest future studies should be RCTs utilising objectively quantified digitised pacing, over a longer duration of examination (i.e. longitudinal studies), using the core outcome set for patient reported outcome measures. Until these are completed, the literature base is insufficient to inform treatment practises for people with ME/CFS and long COVID.

People with Long Covid and ME/CFS Exhibit Similarly Impaired Balance and Physical Capacity: A Case-Case-Control Study.

PURPOSE: Postural sway and physical capacity had not previously been compared between people with long COVID and people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Therefore, this study determined postural sway and physical capacity in people with long COVID (∼16-month illness duration; n = 21) and ME/CFS (∼16-year illness duration; n = 20), vs age-matched healthy controls (n = 20). METHODS: Postural sway was during a 30-s static stand test. Physical capacity was determined using the Timed Up and Go test and 5 Times Sit to Stand test. Throughout, participants wore isoinertial measurement units. RESULTS: Postural sway was worse (ie, greater) in people with long COVID and ME/CFS than controls, but not different between long COVID and ME/CFS. Performance of the Timed Up and Go test and 5 Times Sit to Stand test were worse in long COVID and ME/CFS than controls, but not different between long COVID and ME/CFS. Of long COVID and ME/CFS participants, 87% and 13% exceeded the threshold for muscle weakness in the 5 Times Sit to Stand test and Timed Up and Go test, respectively. CONCLUSIONS: These data suggest that both people with long COVID and people with ME/CFS have similarly impaired balance and physical capacity. Therefore, there is an urgent need for interventions to target postural sway and physical capacity in people with ME/CFS, and given the current pandemic, people with long COVID.

A Cross-Sectional Study of Symptom Prevalence, Frequency, Severity, and Impact of Long-COVID in Scotland: Part I.

BACKGROUND: Commonly reported symptoms of long COVID may have different patterns of prevalence and presentation across different countries. While some limited data have been reported for the United Kingdom, national specificity for Scotland is less clear. We present a cross-sectional survey to examine the symptom prevalence, frequency, and severity of long COVID for people living with the condition in Scotland. METHODS: An online survey was created in the English language and was available between April 21, 2022 and August 5, 2022. Participants were included if they were ≥18 years old, living in Scotland, and had self-diagnosed or confirmed long COVID; and excluded if they were hospitalized during their initial infection. Within this article we quantify symptom prevalence, frequency, severity, and duration. RESULTS: Participants (n = 253) reported the most prevalent long-COVID symptoms to be post-exertional malaise (95%), fatigue/tiredness (85%), and cognitive impairment (68%). Fatigue/tiredness, problems with activities of daily living (ADL), and general pain were most frequently occurring, while sleep difficulties, problems with ADL, and nausea were the most severe. Scottish Index of Multiple Deprivation associated with symptom number, severity, and frequency, whereas vaccine status, age, sex, and smoking status had limited or no association. CONCLUSIONS: These findings outline the challenges faced for those living with long COVID and highlight the need for longitudinal research to ascertain a better understanding of the condition and its longer-term societal impact.

A Cross-Sectional Study of Symptom Prevalence, Frequency, Severity, and Impact of Long COVID in Scotland: Part II.

BACKGROUND: There has been some effort to map the prevalence, frequency, and severity of symptoms of long COVID at local and national levels. However, what is frequently absent from such accounts is details of the impact the disease and its symptoms have had on those living with the condition. In this article, we present details of the impact on work, caring, and mental health gathered using a cross-sectional survey. METHODS: Data were collected using an online survey that was available from April 21, 2022, to August 5, 2022. Included participants had either self-diagnosed or confirmed long COVID, were living in Scotland, and were aged ≥18 years. Hospitalization during initial COVID-19 infection was an exclusion criterion. Participants were asked to report on the impact of their illness on everyday activities such as working, studying, or caring. They also completed an assessment of their current mood. RESULTS: People with long COVID were often severely impacted in their ability to work and study. Severe impact on work and study were predicted by more severe and more frequent fatigue, more severe pain, and more severe cognitive impairment. Respondents' ability to care for child dependents was also associated with more severe and more frequent fatigue, and more severe cognitive impairments. More severe pain associated with greater impact on adult care. Negative mood correlated most strongly with frequency and severity of neurological symptoms, including lack of attention, loss of smell, impaired sense of smell, loss of taste, impaired sense of taste, and loss of appetite. CONCLUSIONS: Long COVID has a significant impact on ability to work, study, and care for dependents. The severity of this impact is associated with specific symptom burden, including fatigue, pain, and cognitive impairment.

Ex Vivo treatment of coronary artery endothelial cells with serum post-exercise training offers limited protection against in vitro exposure to FEC-T chemotherapy.

Background: Chemotherapy treatment for breast cancer associates with well-documented cardiovascular detriments. Exercise has shown promise as a potentially protective intervention against cardiac toxicity. However, there is a paucity of evidence for the benefits of exercise on the vasculature. Objectives: This study aimed to determine the effects of chemotherapy on the vascular endothelium; and if there are protective effects of serological alterations elicited by an exercise training intervention. Methods and Results: 15 women participated in a 12-week home-based exercise intervention consisting of three high-intensity interval sessions per week. Human coronary artery endothelial cells (HCAEC) were exposed to physiological concentrations of 5-fluorouracil, epirubicin, cyclophosphamide (FEC) and docetaxel to determine a dose-response. Twenty-4 hours prior to FEC and docetaxel exposure, HCAECs were preconditioned with serum collected pre- and post-training. Annexin V binding and cleaved caspase-3 were assessed using flow cytometry and wound repair by scratch assays. Chemotherapy exposure increased HCAEC Annexin V binding, cleaved caspase-3 expression in a dose-dependent manner; and inhibited wound repair. Compared to pre-training serum, conditioning HCAECs with post-training serum, reduced Annexin V binding (42% vs. 30%, p = 0.01) when exposed to FEC. For docetaxel, there were no within-group differences (pre-vs post-exercise) for Annexin V binding or cleaved caspase-3 expression. There was a protective effect of post-training serum on wound repair for 5-flurouracil (p = 0.03) only. Conclusion: FEC-T chemotherapy drugs cause significant damage and dysfunction of endothelial cells. Preconditioning with serum collected after an exercise training intervention, elicited some protection against the usual toxicity of FEC-T, when compared to control serum.

High-Intensity Interval Training (HIIT) in Hypoxia Improves Maximal Aerobic Capacity More Than HIIT in Normoxia: A Systematic Review, Meta-Analysis, and Meta-Regression.

The present study aimed to determine the effect of high intensity interval training (HIIT) in hypoxia on maximal oxygen uptake (VO2max) compared with HIIT in normoxia with a Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)-accordant meta-analysis and meta-regression. Studies which measured VO2max following a minimum of 2 weeks intervention featuring HIIT in hypoxia versus HIIT in normoxia were included. From 119 originally identified titles, nine studies were included (n = 194 participants). Meta-analysis was conducted on change in (∆) VO2max using standardised mean difference (SMD) and a random effects model. Meta-regression examined the relationship between the extent of environmental hypoxia (fractional inspired oxygen [FiO2]) and ∆VO2max and intervention duration and ∆VO2max. The overall SMD for ∆VO2max following HIIT in hypoxia was 1.14 (95% CI = 0.56-1.72; p < 0.001). Meta-regressions identified no significant relationship between FiO2 (coefficient estimate = 0.074, p = 0.852) or intervention duration (coefficient estimate = 0.071, p = 0.423) and ∆VO2max. In conclusion, HIIT in hypoxia improved VO2max compared to HIIT in normoxia. Neither extent of hypoxia, nor training duration modified this effect, however the range in FiO2 was small, which limits interpretation of this meta-regression. Moreover, training duration is not the only training variable known to influence ∆VO2max, and does not appropriately capture total training stress or load. This meta-analysis provides pooled evidence that HIIT in hypoxia may be more efficacious at improving VO2max than HIIT in normoxia. The application of these data suggest adding a hypoxic stimuli to a period of HIIT may be more effective at improving VO2max than HIIT alone. Therefore, coaches and athletes with access to altitude (either natural or simulated) should consider implementing HIIT in hypoxia, rather than HIIT in normoxia where possible, assuming no negative side effects.

Breast cancer chemotherapy vascular toxicity: a review of mediating mechanisms and exercise as a potential therapeutic.

Breast cancer chemotherapy, although very potent against tumour tissue, results in significant cardiovascular toxicity. The focus of research in this area has been predominantly towards cardiotoxicity. There is limited evidence detailing the impact of such treatment on the vasculature despite its central importance within the cardiovascular system and resultant detrimental effects of damage and dysfunction. This review highlights the impact of chemotherapy for breast cancer on the vascular endothelium. We consider the most likely mechanisms of endothelial toxicity to be through direct damage and dysfunction of the endothelium. There are sharp consequences of these detrimental effects as they can lead to cardiovascular disease. However, there is potential for exercise to alleviate some of the vascular toxicity of chemotherapy, and the evidence for this is provided. The potential role of exercise in protecting against vascular toxicity is explained, highlighting the recent in-human and animal model exercise interventions. Lastly, the mediating mechanisms of exercise protection of endothelial health is discussed, focusing on the importance of exercise for endothelial health, function, repair, inflammation and hyperlipidaemia, angiogenesis, and vascular remodelling. These are all important counteracting measures against chemotherapy-induced toxicity and are discussed in detail.

Improving management of comorbidity in patients with colorectal cancer using comprehensive medical assessment: a pilot study.

BACKGROUND: Screening for and active management of comorbidity soon after cancer diagnosis shows promise in altering cancer treatment and outcomes for comorbid patients. Prior to a large multi-centre study, piloting of the intervention (comprehensive medical assessment) was undertaken to investigate the feasibility of the comorbidity screening tools and proposed outcome measures, and the feasibility, acceptability and potential effect of the intervention. METHODS: In this pilot intervention study, 72 patients of all ages (36 observation/36 intervention) with newly diagnosed or recently relapsed colorectal adenocarcinoma were enrolled and underwent comorbidity screening and risk stratification. Intervention patients meeting pre-specified comorbidity criteria were referred for intervention, a comprehensive medical assessment carried out by geriatricians. Each intervention was individually tailored but included assessment and management of comorbidity, polypharmacy, mental health particularly depression, functional status and psychosocial issues. Recruitment and referral to intervention were tracked, verbal and written feedback were gathered from staff, and semi-structured telephone interviews were conducted with 13 patients to assess screening tool and intervention feasibility and acceptability. Interviews were transcribed and analysed thematically. Patients were followed for 6-12 months after recruitment to assess feasibility of proposed outcome measures (chemotherapy uptake and completion rates, grade 3-5 treatment toxicity, attendance at hospital emergency clinic, and unplanned hospitalisations) and descriptive data on outcomes collated. RESULTS: Of the 29 intervention patients eligible for the intervention, 21 received it with feedback indicating that the intervention was acceptable. Those in the intervention group were less likely to be on 3+ medications, to have been admitted to hospital in previous 12 months, or to have limitations in daily activities. Collection of data to measure proposed outcomes was feasible with 55% (6/11) of intervention patients completing chemotherapy as planned compared to none (of 14) of the control group. No differences were seen in other outcome measures. Overall the study was feasible with modification, but the intervention was difficult to integrate into clinical pathways. CONCLUSIONS: This study generated valuable results that will be used to guide modification of the study and its approaches prior to progressing to a larger-scale study. TRIAL REGISTRATION: Retrospective, 26 August 2019, ACTRN12619001192178.

Case Report of Exercise to Attenuate Side Effects of Treatment for Pancreatic Cancer.

Treatments for pancreatic cancer can have debilitating side effects including fatigue, weight loss, and cardiac toxicity, resulting in functional loss and psychological distress. Exercise has been proposed as a therapy to counteract physical and psychological detriments. The case: A 47-year-old male undergoing chemotherapy for stage 3 locally advanced pancreatic cancer. He was cycling during hospital chemotherapy infusions (6 fortnightly cycles of FOLFIRINOX: 5-FU 2, 400 mg/m2, over 48 h: irinotecan 180 mg/m2, oxaliplatin 85 mg/m2, no 5-FU bolus) plus 12 weeks of twice weekly aerobic and resistance exercise. Over 12 weeks, body composition was maintained, and physical function improved, with specific increases in muscular strength of up to 50% and aerobic capacity improving by 9%. Moreover, quality of life, fatigue, psychological distress, and sleep quality improved by 38, 113, 50, and 9%, respectively. Additionally, the participant experienced more severe side effects in week 6, when he did not cycle to a high intensity during hospital infusion and had less total weekly exercise. After cycle 6 (week 11), chemotherapy was halted, and a Whipple resection procedure was successfully performed. It can be concluded that regular aerobic and resistance exercise plus exercise during infusion can attenuate expected decline in physical and mental health with pancreatic cancer treatment and may reduce treatment side effects and have favourable effects on prognosis.

Extracellular Localisation of the C-Terminus of DDX4 Confirmed by Immunocytochemistry and Fluorescence-Activated Cell Sorting.

Putative oogonial stem cells (OSCs) have been isolated by fluorescence-activated cell sorting (FACS) from adult human ovarian tissue using an antibody against DEAD-box helicase 4 (DDX4). DDX4 has been reported to be germ cell specific within the gonads and localised intracellularly. White et al. (2012) hypothesised that the C-terminus of DDX4 is localised on the surface of putative OSCs but is internalised during the process of oogenesis. This hypothesis is controversial since it is assumed that RNA helicases function intracellularly with no extracellular expression. To determine whether the C-terminus of DDX4 could be expressed on the cell surface, we generated a novel expression construct to express full-length DDX4 as a DsRed2 fusion protein with unique C- and N-terminal epitope tags. DDX4 and the C-terminal myc tag were detected at the cell surface by immunocytochemistry and FACS of non-permeabilised human embryonic kidney HEK 293T cells transfected with the DDX4 construct. DDX4 mRNA expression was detected in the DDX4-positive sorted cells by RT-PCR. This study clearly demonstrates that the C-terminus of DDX4 can be expressed on the cell surface despite its lack of a conventional membrane-targeting or secretory sequence. These results validate the use of antibody-based FACS to isolate DDX4-positive putative OSCs.

Characterization of follicles in girls and young women with Turner syndrome who underwent ovarian tissue cryopreservation.

OBJECTIVE: To characterize ovarian follicles of girls and young women with Turner syndrome (TS) who underwent ovarian tissue cryopreservation (OTC). DESIGN: Retrospective case-control study. SETTING: University hospital. PATIENT(S): Fifteen girls and young women with TS aged 5-22 years at OTC were included, together with 42 control girls and young women aged 1-25 years who underwent OTC because of cancer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle density (follicles/mm3), morphology, and health were assessed in ovarian cortex biopsies from TS patients and compared with controls. Hormone concentrations were measured in serum and follicle fluids. Immature cumulus oocyte complexes were obtained and matured in vitro. RESULT(S): Follicles were found in 60% of the biopsies (9 of 15) from TS ovaries. In 78% of the ovaries (7 of 9) with follicles, the follicle density was within the 95% confidence interval of the control group. There was a high rate of abnormal follicle morphology. Six follicle-specific proteins were expressed similarly in TS and control ovaries. However, apoptosis and zona pellucida protein expression were found to be abnormal in TS. Turner syndrome follicle fluid from small antral follicles had lower concentrations of estrogen and testosterone and higher concentrations of antimüllerian hormone than controls. Thirty-one cumulus oocyte complexes were collected from one patient and cultured for 48 hours in vitro, resulting in five metaphase II oocytes (maturation rate 16%, degeneration rate 19%). CONCLUSION(S): The benefits of OTC may be limited to a highly selected group of TS mosaic patients in whom a sizeable pool of normal follicles is present at OTC.

Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response.

STUDY QUESTION: Does ovarian follicle activation by phosphatase homologue of chromosome-10 (PTEN) inhibition affect DNA damage and repair in bovine oocytes and granulosa cells? SUMMARY ANSWER: PTEN inhibition promotes bovine non-growing follicle activation but results in increased DNA damage and impaired DNA repair capacity in ovarian follicles in vitro. WHAT IS KNOWN ALREADY: Inhibition of PTEN is known to activate primordial follicles but may compromise further developmental potential. In breast cancer cells, PTEN inhibition represses nuclear translocation of breast cancer susceptibility 1 (BRCA1) and Rad51; this impairs DNA repair resulting in an accumulation of damaged DNA, which contributes to cell senescence. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue fragments were exposed to control medium alone or containing either 1 or 10 µM bpv(HOpic), a pharmacological inhibitor of PTEN, in vitro for 24 h. A sub-group of tissue fragments were collected for Western blot analysis after bpv(HOpic) exposure. The remainder were incubated in control medium for a further 5 days and then analysed histologically and by immunohistochemistry to detect DNA damage and repair pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine ovaries were obtained from abattoir-slaughtered heifers. Tissue fragments were exposed to either control medium alone or medium containing either 1 µM or 10 µM bpv(HOpic) for 24 h. Tissue fragments collected after 24 h were subjected to Akt quantification by Western blotting (six to nine fragments per group per experiment). Follicle stage and morphology were classified in remaining fragments. Immunohistochemical analysis included nuclear exclusion of FOXO3 as a marker of follicle activation, γH2AX as a marker of DNA damage, meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM), Rad51, breast cancer susceptibility 1 (BRCA1) and breast cancer susceptibility 2 (BRCA2) as DNA repair factors. A total of 29 550 follicles from three independent experiments were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Tissue fragments exposed to bpv(HOpic) had increased Akt phosphorylation at serine 473 (pAkt/Akt ratio, 2.25- and 6.23-fold higher in 1 and 10 µM bpv(HOpic) respectively compared to control, P < 0.05). These tissue fragments contained a significantly higher proportion of growing follicles compared to control (78.6% in 1 µM and 88.7% in 10 µM versus 70.5% in control; P < 0.001). The proportion of morphologically healthy follicles did not differ significantly between 1 µM bpv(HOpic) and control (P < 0.001) but follicle health was lower in 10 µM compared to 1 µM and control in all follicle types (P < 0.05). DNA damage in oocytes, indicated by expression of γH2AX, increased following exposure to 1 µM bpv(HOpic) (non-growing, 83%; primary follicles, 76%) and 10 µM (non-growing, 77%; primary, 84%) compared to control (non-growing, 30% and primary, 59%) (P < 0.05 for all groups). A significant reduction in expression of DNA repair proteins MRE11, ATM and Rad51 was observed in oocytes of non-growing and primary follicles of treatment groups (primary follicles in controls versus 10 µM bpv(HOpic): MRE, 68% versus 47%; ATM, 47% versus 18%; Rad51, 48% versus 24%), P < 0.05 for all groups. Higher dose bpv(HOpic) also resulted in lower expression of BRCA1 compared to control and 1 µM bpv(HOpic) (P < 0.001) in non-growing and primary follicles. BRCA2 expression was increased in oocytes of primary follicles in 1 µM bpv(HOpic) (36%) compared to control (20%, P = 0.010) with a marked decrease in 10 µM (1%, P ≤ 0.001). Granulosa cells of primary and secondary follicles in bpv(HOpic) groups showed more DNA damage compared to control (P < 0.05). However, bpv(HOpic) did not impact granulosa cell DNA repair capacity in secondary follicles, but BRCA1 declined significantly in higher dose bpv(HOpic). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study focuses on non-growing follicle activation after 6 days culture and may not reflect DNA damage and repair capacity in later stages of oocyte and follicle growth. WIDER IMPLICATIONS OF THE FINDINGS: In vitro activation of follicle growth may compromise the bidirectional signalling between oocyte and granulosa cells necessary for optimal oocyte and follicle health. This large animal model may be useful in optimising follicle activation protocols with a view to transfer for clinical application. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Indonesia endowment fund for education. No competing interest. TRIAL REGISTRATION NUMBER: Not applicable.

In vitro growth of immature bovine follicles and oocytes.

The limitation in the supply of mature, fertilisable oocytes constitutes a major impediment to increasing the success of assisted reproduction, stem cell derivation and cloning in domestic species. Techniques are being developed to grow immature oocytes invitro that have the potential to increase the supply of oocytes. Mouse oocytes can be cultured from initial stages of development to maturity, and live young have been produced, but for domestic species, such as cows, with long growth periods, invitro systems that allow complete growth of oocytes contained within primordial follicles to maturity is technically challenging and has not yet been achieved. For cows, several culture systems have been developed that support specific developmental stages, but a multistep culture system will be required for complete growth invitro. This review highlights the steps that will be required to achieve the goal of growing oocytes invitro.

Initial characterisation of adult human ovarian cell populations isolated by DDX4 expression and aldehyde dehydrogenase activity.

The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.

The controversial existence and functional potential of oogonial stem cells.

The regenerative potential of the mammalian ovary has been a controversial area over the last decade. Isolation of cells, termed oogonial stem cells (OSCs), from adult rodent and human ovaries has been reported, with these cells exhibiting both germ and stem cell markers in culture. When re-introduced into an ovarian somatic environment these cells have generated follicles capable of producing healthy offspring in rodents, and there is some evidence of human OSCs being able to form oocyte-like structures in a xenotransplant model. Importantly, there are no data on their potential physiological role within the ovary, and specifically no evidence that they contribute to the primordial follicle pool and thus to later stages of follicle development. The cues required for oocyte differentiation from these cells are not well understood either in vivo or in vitro, and these will need to be further elucidated to maximise their potential for therapeutic intervention. OSCs may also be of value as a model to investigate normal human germ cell differentiation. It is likely that their interactions with ovarian somatic cells and/or extracellular signals will be important in these processes. This review summarises our current knowledge on the isolation and characterisation of mammalian oogonial stem cells.

Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles.

In the mammalian ovary a small number of follicles are steadily recruited from the quiescent pool to undergo development. Follicle loss, maintenance and growth are strictly controlled by complex molecular interactions including the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway. Stimulation of PI3K promotes phosphorylation of Akt resulting in follicle survival and activation of growth whereas this pathway is suppressed by the actions of the phosphatase and tensin homologue (PTEN). The aim of this study was to determine the effect of dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (bpV), a reversible inhibitor of PTEN, on the activation, survival and development of human ovarian follicles in vitro. Biopsied ovarian tissue fragments were obtained from 17 women aged 23-46 years and exposed to 1 µM bpV(HOpic) (n = 146) or control medium (n = 128) for 24 h. Media were then replaced with control medium and all tissue incubated for a further 5 days. Ovarian tissue from each treatment group was fixed after the initial 24 h culture period and phosphorylated Akt was quantified by western blotting. After 6 days incubation all tissue fragments were inspected under light microscopy and any secondary follicles ≥100 µm isolated. Isolated follicles were cultured individually in control medium supplemented with 100 ng/ml recombinant human activin A. Tissue fragments without follicles suitable for isolation were fixed and processed for histological and immunohistochemical analysis. During 6 days culture, follicle activation occurred in tissue samples from both treatment groups but with significantly more follicles progressing to the secondary stage of development in the presence of 1 µM bpV(HOpic) compared with control (31 versus 16%; P < 0.05). Increased activation was associated with increased Akt phosphorylation and increased nuclear export of FOXO3. However isolated and cultured follicles that had been exposed to bpV(HOpic) showed limited growth and reduced survival compared with follicles from control fragments (P < 0.05). This study demonstrates that inhibition of PTEN with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles.