Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

STUDY QUESTION: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? SUMMARY ANSWER: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. WHAT IS KNOWN ALREADY: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-µM gallic acid on their maturation rate. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/ß-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. LARGE SCALE DATA: Raw data from this study can be accessed through GSE158539. LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Post-fracture care gap: a retrospective population-based analysis of Hong Kong from 2009 to 2012.

INTRODUCTION: Patients who sustain an osteoporotic fracture are at increased risk of sustaining further osteoporotic fracture. The risk can be reduced by prescription of anti-osteoporosis medication. The aim of the present study was to determine the current practice in Hong Kong regarding secondary drug prevention of fragility fractures after osteoporotic hip fracture. METHODS: Dispensation of anti-osteoporosis medication records from patients with new fragility hip fractures aged ≥65 years were retrieved using the Hospital Authority Clinical Data Analysis and Reporting System from 2009 to 2012. The intervention rate each year was determined from the percentage of patients receiving anti-osteoporosis medication within 1 year after hip fracture. RESULTS: A total of 15 866 patients with osteoporotic hip fracture who met the criteria were included. The intervention rate differed each year from 2009 to 2012, ranging between 9% and 15%. Orthopaedic surgeons initiated 63% of anti-osteoporosis medication, whereas physicians initiated 37%. The anti-osteoporosis drugs being prescribed included alendronic acid (76%), ibandronic acid (12%), strontium ranelate (5%), and zoledronic acid (4%). CONCLUSION: Most patients with hip fracture remained untreated for 1 year after the osteoporotic hip fracture. The Hospital Authority should allocate more resources to implement a best practice framework for treatment of patients with hip fracture at high risk of secondary fracture.

Operative outcome of Hong Kong centenarians with hip fracture.

INTRODUCTION: International clinical guidelines recommend early surgical treatment for geriatric patients with hip fracture. There are, however, few data concerning the operative outcome of centenarians. This study aimed to report the epidemiology of hip fracture and postoperative mortality rate, and to discuss whether operation is justified in centenarians in Hong Kong. METHODS: This observational study was carried out in all public hospitals of Hong Kong. All patients aged 100 years or above who underwent hip fracture surgery in any public hospital between 1 January 2010 and 31 December 2013 were included. Their postoperative mean and median survival time was recorded. RESULTS: Of 114 centenarians, 96 (84%) were female. The age of patients ranged from 100 to 109 years, with the largest number (44%) aged 100 years. The follow-up interval ranged from 5 to 1619 days (median, 412 days; interquartile range, 683 days). The 1-month, 6-month, and 1-year mortalities were 8%, 25%, and 37%, respectively. By Kaplan-Meier analysis, the postoperative mean survival was 2 years 2 months (95% confidence interval, 680-936 days) and the median survival time was 2 years (interquartile range, 1234 days). CONCLUSION: The 1-year mortality among Hong Kong centenarians with hip fracture was 37%, which is lower than the 41.1% in the general centenarian population in Japan. The median survival time after hip fracture surgery was 2 years, suggesting that surgery even at an extreme age is worthwhile to maintain quality of life. Extreme age should not be a barrier to operative treatment.

Improvement in nitrogen fixation capacity could be part of the domestication process in soybean.

Biological nitrogen fixation (BNF) in soybeans is a complex process involving the interplay between the plant host and the symbiotic rhizobia. As nitrogen supply has a crucial role in growth and development, higher nitrogen fixation capacity would be important to achieve bigger plants and larger seeds, which were important selection criteria during plant domestication by humans. To test this hypothesis, we monitored the nitrogen fixation-related performance in 31 cultivated and 17 wild soybeans after inoculation with the slow-growing Bradyrhizobium diazoefficiens sp. nov. USDA110 and the fast-growing Sinorhizobium (Ensifer) fredii CCBAU45436. Our results showed that, in general, cultivated soybeans gave better performance in BNF. Electron microscopic studies indicated that there was an exceptionally high accumulation of poly-ß-hydroxybutyrate bodies in bacteroids in the nodules of all wild soybeans tested, suggesting that the C/N balance in wild soybeans may not be optimized for nitrogen fixation. Furthermore, we identified new quantitative trait loci (QTLs) for total ureides and total nodule fresh weight by employing a recombinant inbred population composed of descendants from a cross between a cultivated and a wild parent. Using nucleotide diversity (θπ), divergence index (Fst) and distribution of fixed single-nucleotide polymorphisms as parameters, we found that some regions in the total ureides QTL on chromosome 17 and the total nodule fresh weight QTL on chromosome 12 exhibited very low diversity among cultivated soybeans, suggesting that these were traits specially selected during the domestication and breeding process.

Effect of disease activity and damage on quality of life in patients with systemic lupus erythematosus: a 2-year prospective study.

OBJECTIVES: To examine the effect of disease activity and damage on health-related quality of life (HRQoL) in patients with systemic lupus erythematosus (SLE). METHODS: Consecutive SLE patients and matched controls were recruited for a study of HRQoL using the Medical Outcomes Study Short Form-36 (SF-36). SLE activity and damage was assessed by the Safety of Oestrogens in Lupus Erythematosus National Assessment SLE Disease Activity Index (SELENA-SLEDAI) and the American College of Rheumatology/Systemic Lupus International Collaborating Clinics (ACR/SLICC) Damage Index (SDI), respectively. Patients were prospectively followed for repeat HRQoL assessment at 2 years. The effects of cumulative disease activity and new damage on changes in SF-36 scores were evaluated. RESULTS: One hundred and fifty-five patients were studied (94% women; age 37.8+/-11.3 years; SLE duration 7.2+/-5.4 years). Fifty (32%) patients had active disease and 75 (48%) had organ damage at baseline. Compared with age- and gender-matched controls, SLE patients had lower SF-36 scores, and the difference remained significant after adjustment for income and education level. SF-36 scores in SLE patients correlated inversely with SDI but not with SELENA-SLEDAI scores. After 2 years, there was a significant drop in the mental component score of the SF-36. Regression analysis revealed that new damage was the only determinant for a reduction in SF-36 scores. Patients with higher cumulative disease activity had a greater drop in bodily pain and general health subscores. CONCLUSIONS: Impaired HRQoL is more common in SLE patients than controls, regardless of age, sex, education and poverty. Pre-existing organ damage is associated with poorer HRQoL and new damage predicts a further decline in HRQoL. Persistent disease activity is associated with deterioration in certain domains of the SF-36.

Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Abnormal albuminuria as a predictor of mortality and renal impairment in Chinese patients with NIDDM.

OBJECTIVE: Microalbuminuria predicts mortality in non-insulin-dependent diabetes mellitus (NIDDM), but its association with deterioration of renal function remains more controversial than in insulin-dependent diabetes mellitus (IDDM). Using albumin-to-creatinine ratios (ACRs) in random spot urine samples is a convenient method for evaluating albuminuria. We studied prospectively the predictive values of albuminuria in NIDDM when assessed by this urine measurement. RESEARCH DESIGN AND METHODS: Between 1991 and 1992, we restudied the clinical and biochemical status of 403 Chinese NIDDM patients recruited in 1989 after a follow-up period of 26.6 +/- 3.2 months (mean +/- SD). Spot urine ACR was measured on two occasions and microalbuminuria was defined as a mean ACR between 5.6 and 38 mg/mmol. RESULTS: From the original cohort, 29 patients had died mostly because of cardiovascular events with or without renal failure. The overall relative risk of death in patients with abnormal albuminuria was 7.1 (P < 0.001) (microalbuminuria: 3.7, P = 0.04; macroalbuminuria: 11, P < 0.001). On multivariate analysis, the independent predictive factors for mortality were plasma creatinine (wald = 12.1, P < 0.001) and glucose concentrations (wald = 10.4, P < 0.001) in the normo- and microalbuminuric patients (n = 11) and age (wald = 4.4, P = 0.03) and plasma creatinine (wald = 8.2, P < 0.01) in the macroalbuminuric group (n = 18). In the survivors (n = 374), baseline spot urine ACR was independently associated with 2-year spot urine ACR in the normo- (P < 0.001), micro- (P < 0.01), and macroalbuminuric groups (P = 0.01). In addition, baseline spot urine ACR was independently related to 2-year plasma creatinine (P = 0.01) in the macroalbuminuric group. The rates of change of the reciprocal of plasma creatinine ( delta [Cr]-1) were -27.3 +/- 62.5, -43.4 +/- 68.6, and -108.8 +/- 98.81.mumol01.month-1 in the normo-, micro-, and macroalbuminuric groups, respectively (P < 0.001). The delta [Cr]-1 was independently and inversely related to the baseline spot urine ACR (P < 0.001) and 2-year systolic blood pressure (P < 0.001). CONCLUSIONS: Abnormal albuminuria as indicated by a random spot urine ACR > 5.6 mg/mmol predicts increased mortality and is associated with the progression of albuminuria and deterioration of renal function in Chinese NIDDM patients.

Mandibular osteomyelitis caused by Blastoschizomyces capitatus in a child with acute myelogenous leukemia.

A 6-year-old girl with acute myelogenous leukemia (AML) developed fungal mandibular osteomyelitis during chemotherapy. Blastoschizomyces capitatus was recognized histologically by its yeast-like morphology and formation of annelloconidia, and was confirmed by culture. The fungal osteomyelitis of the mandible was treated successfully with prolonged antifungal medication, extensive surgical debridement and an oral care program, without interrupting leukemia chemotherapy. B. capitatus osteomyelitis of the mandible may occur during chemotherapy in AML patients with poor dental condition. Successful treatment can be achieved by careful management without interruption of antineoplastic chemotherapy.

Risk and predictors of work disability in Chinese patients with systemic lupus erythematosus.

The aim of this study is to determine the risk and predictive factors for work disability in patients with SLE. A cross-sectional questionnaire study was performed to evaluate the employment status of a sample of consecutive Chinese patients with SLE. Demographic, socioeconomic data (age, gender, marital status, years of education and household income), employment status, self-reported fatigue score and disease characteristics (SLE duration, organ damage and disease activity) were collected. Work disability was defined by the failure to work due to SLE. The cumulative incidence of work disability since the time of SLE diagnosis was studied by a Kaplan Meier's plot, and factors predictive of work disability were studied by Cox regression. A total of 147 patients with SLE were studied (mean age = 39.4 +/- 11.3 years; 95% women). Among 105 patients who were working at the time of SLE diagnosis, 39 (37%) lost their ability to work as a result of SLE after a mean disease duration of 10.0 +/- 6.1 years. Twenty-two (56%) patients lost their work ability within 2 years of diagnosis of SLE. The self-reported reasons for job loss were musculoskeletal pain (87%), skin disease (26%), renal problem (21%), fatigue (85%), memory deterioration (51%), anxiety or depressive symptoms (74%), too frequent sick leave (10%) and long-term hospitalisation (10%). The cumulative risk of work disability was 36% at 5 years after SLE diagnosis. In a Cox regression model, age (HR = 1.06 [1.02-1.11] per year; P = 0.008), self-reported fatigue score (HR = 1.06 [1.01-1.10] per point; P = 0.01) and mean disease activity score in the preceding two years (HR = 1.20 [1.02-1.42] per point; P = 0.03) were independently associated with working disability. In all, 37% of this group of patients with SLE lost their work ability after having the disease for 10 years. More than 50% of these patients developed work disability within the first 2 years of SLE diagnosis. Older age, fatigue and more active disease were independent predictors of work disability.

The impact of two different malocclusion traits on quality of life.

OBJECTIVES: The purpose of this prospective study was to assess the impact of two occlusal traits on the quality of life of children and their families. METHODS: A total of 180 subjects, which included 90 consecutive patients (aged 13-15 years) and their parents, were recruited on the basis of predetermined criteria to the following groups: increased overjet, spaced dentition and control. Each subject and their parent underwent separate supervised completion of a Child or Parental-Caregiver Perception questionnaire, respectively, which are components of the Child Oral Health Quality of Life questionnaire. RESULTS: The three groups were shown not to demonstrate any differences in socio-demographic characteristics. Statistically significant differences were observed between children in the control group and their counterparts in the increased overjet (p = 0.002) and spaced dentition (p < 0.001) groups. However, no such difference was detected between children in the increased overjet and spaced dentition groups (p = 0.5). Parents of these children demonstrated similar statistical findings: p = 0.007, p = 0.003 and p = 0.9, respectively. CONCLUSIONS: Occlusal traits such as an increased overjet and a spaced dentition have a significant negative impact on both the children's and their families' quality of life.

Intraperitoneal bleeding from ectopic decidua following hormonal contraception. Case report.

PIP: This article reports the case of a bleeding ectopic decidual reaction from the serosal surface of the fallopian tube in the absence of pregnancy in a woman who had received a long-acting injectable contraceptive. This complication has not been previously reported as a possible side-effect of progestogen administration. The patient, a 42-year old Chinese woman, received 3 injections of medroxyprogesterone acetate 150 mg at 3-month intervals and then switched to the combined contraceptive Microgynon (.15 mg levonorgestrel and .03 mg ethinyl estradiol). The 2nd course of Microgynon was completed 1 week before her admission to the hospital with right lower abdominal pain and secondary amenorrhea for 9 months. A hematocele was found on the right side of the pouch of Douglas. Decidual reaction is an exaggeration of the normal response of the endometrium to progesterone. In the absence of pregnancy, ectopic decidual changes have been attributed to the stimulation of appropriate cells by progesterone and progesterone-like substances from the corpus luteum or the adrenal cortex.^ieng

Long-term effects of angiotensin-converting enzyme inhibition and metabolic control in hypertensive type 2 diabetic patients.

UNLABELLED: Long-term effects of angiotensin-converting enzyme inhibition and metabolic control in hypertensive type 2 diabetic patients. BACKGROUND: In hypertensive type 2 diabetic patients, treatment with angiotensin-converting enzyme (ACE) inhibitors is associated with a lower incidence of cardiovascular events than those treated with calcium channel-blocking agents. However, the long-term renal effects of ACE inhibitors in these patients remain inconclusive. In 1989, we commenced a placebo-controlled, double-blind, randomized study to examine the anti-albuminuric effects of enalapril versus nifedipine (slow release) in 102 hypertensive, type 2 diabetic patients. These patients have been followed up for a mean trial duration of 5.5 +/- 2.2 years. We examined the determinants, including the effect of ACE inhibition on clinical outcomes in these patients. METHODS: After a six-week placebo-controlled, run-in period, 52 patients were randomized double-blind to receive nifedipine (slow release) and 50 patients to receive enalapril. After the one-year analysis, which confirmed the superior anti-albuminuric effects of enalapril (-54%) over nifedipine (+11%), all patients were continued on their previously assigned treatment with informed consent. They were subdivided into normoalbuminuric (N = 43), microalbuminuric (N = 34), and macroalbuminuric (N = 25) groups based on two of three 24-hour urinary albumin excretion (UAE) measurements during the run-in period. Renal function was shown by the 24-hour UAE, creatinine clearance (CCr), and the regression coefficient of the yearly plasma creatinine reciprocal (beta-1/Cr). Clinical endpoints were defined as death, cardiovascular events, and/or renal events (need for renal replacement therapy or doubling of baseline plasma creatinine). RESULTS: In the whole group, patients treated with enalapril were more likely to revert to being normoalbuminuric (23.8 vs. 15.4%), and fewer of them developed macroalbuminuria (19.1 vs. 30.8%) compared with the nifedipine-treated patients (P < 0.05). In the microalbuminuric group, treatment with enalapril (N = 21) was associated with a 13.0% (P < 0.01) reduction in 24-hour UAE compared with a 17.3% increase in the nifedipine group (N = 13). In the macroalbuminuric patients, enalapril treatment (N = 11) was associated with stabilization compared with a decline in renal function in the nifedipine group, as shown by the beta-1/Cr (0.65 +/- 4.29 vs. -1.93 +/- 2.35 1/micromol x 10-3, P < 0.05) after adjustment for baseline values. Compared with the normoalbuminuric and microalbuminuric patients, those with macroalbuminuria had the lowest mean CCr (75.5 +/- 24.1 vs. 63.5 +/- 21.3 vs. 41.9 +/- 18.5 mL/min, P < 0.001) and the highest frequency of clinical events (4.7 vs. 5.9 vs. 52%, P < 0. 001). On multivariate analysis, beta-1/Cr (R2 = 0.195, P < 0.001) was independently associated with baseline HbA1c (beta = -0.285, P = 0.004), whereas clinical outcomes (R2 = 0.176, P < 0.001) were independently related to the mean low-density lipoprotein cholesterol (beta = 2.426, P = 0.018), high-density lipoprotein cholesterol (beta = -8.797, P = 0.03), baseline UAE (beta = 0.002, P = 0.04), and mean CCr during treatment (beta = -0.211, P = 0.006). CONCLUSION: In this prospective cohort analysis involving 102 hypertensive, type 2 diabetic patients with varying degrees of albuminuria followed up for a mean duration of five years, we observed the importance of good metabolic and blood pressure control on the progression of albuminuria and renal function. Treatment with enalapril was associated with a greater reduction in albuminuria than with nifedipine in the entire patient group, and especially in those with microalbuminuria. In the macroalbuminuric patients, the rate of deterioration in renal function was also attenuated by treatment with enalapril.