The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

STUDY QUESTION: What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? SUMMARY ANSWER: Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. WHAT IS KNOWN ALREADY: Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. MAIN RESULTS AND THE ROLE OF CHANCE: The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Progesterone and 17-hydroxy-progesterone concentrations in follicular fluid and serum reflect their production in granulosa and theca cells.

RESEARCH QUESTION: How is the production of progesterone (P4) and 17-hydroxy-P4 (17-OH-P4) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? DESIGN: Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. RESULTS: In the early follicular phase, theca cells primarily synthesized 17-OH-P4 while granulosa cells produced limited P4, maintaining the P4:17-OH-P4 ratio <1. As follicles reached follicle selection at a diameter of approximately 10 mm, P4 synthesis in granulosa cells was up-regulated, but P4 was mainly accumulated in follicular fluid. During final maturation, enhanced activity of the enzyme HSD3B2 in granulosa cells enhanced P4 production, with the P4:17-OH-P4 ratio increasing to >1. The concentration of 17-OH-P4 in the luteal phase was similar to that in the follicular phase, but P4 production increased in the luteal phase, yielding a P4:17-OH-P4 ratio significantly >1. CONCLUSIONS: The P4:17-OH-P4 ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P4 during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

Developmental stage and morphology of the competent blastocyst are associated with sex of the child but not with other obstetric outcomes: a multicenter cohort study.

STUDY QUESTION: Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child? SUMMARY ANSWER: A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed. WHAT IS KNOWN ALREADY: The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting. STUDY DESIGN, SIZE, DURATION: Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833). MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)). LIMITATIONS, REASONS FOR CAUTION: It is possible that some residual confounding remains. WIDER IMPLICATIONS OF THE FINDINGS: Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth. STUDY FUNDING/COMPETING INTEREST(S): Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Follicular hormone dynamics during the midcycle surge of gonadotropins in women undergoing fertility treatment.

Changes in concentrations of intra-follicular hormones during ovulation are important for final oocyte maturation and endometrial priming to ensure reproductive success. As no human studies have investigated these changes in detail, our objective was to describe the dynamics of major follicular fluid (FF) hormones and transcription of steroidogenic enzymes and steroid receptors in human granulosa cells (GCs) during ovulation. We conducted a prospective cohort study at a public fertility clinic in 2016-2018. Fifty women undergoing ovarian stimulation for fertility treatment were included. From each woman, FF and GCs were collected by transvaginal ultrasound-guided follicle puncture of one follicle at two specific time points during ovulation, and the study covered a total of five time points: before ovulation induction (OI), 12, 17, 32 and 36 h after OI. Follicular fluid concentrations of oestradiol, progesterone, androstenedione, testosterone, 17-hydroxyprogesterone, anti-Mullerian hormone, inhibin A and inhibin B were measured using ELISA assays, and a statistical mixed model was used to analyse differences in hormone levels between time points. Gene expression of 33 steroidogenic enzymes and six hormone receptors in GCs across ovulation were assessed by microarray analysis, and selected genes were validated by quantitative reverse transcription PCR. We found that concentrations of oestradiol, testosterone, progesterone, AMH, inhibin A and inhibin B (P < 0.001) and gene expression of 12 steroidogenic enzymes and five receptors (false discovery rate < 0.0001) changed significantly during ovulation. Furthermore, we found parallel changes in plasma hormones. The substantial changes in follicular hormone production during ovulation highlight their importance for reproductive success.

Two waves of transcriptomic changes in periovulatory human granulosa cells.

STUDY QUESTION: How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER: Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY: The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell-matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36 h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. LARGE SCALE DATA: The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. WIDER IMPLICATIONS OF THE FINDINGS: The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.

Perceptions of Professional Responsibility When Caring for Older People in Home Care in Sweden.

Older people in Sweden are increasingly being cared for in the own home, where professional caregivers play an important role. This study aimed to describe perceptions of caring responsibility in the context of older people's homes from the perspective of professional caregivers from caring professions. Fourteen interviews were conducted with professional caregivers from different professions. The result show how professional caregivers perceive responsibility as limitless, constrained by time, moral, overseeing, meaningful and lonesome. Responsibility seems to affect caregivers to a large extent when the burden is high. Professional caregivers' perceptions of responsibility, and the potential consequences of a perceived strained work situation therefore need to be addressed. The findings also indicate a need for professional support and guidance when it is difficult to distinguish between professional and personal responsibility.

Radiofrequency ablation as initial therapy in paroxysmal atrial fibrillation: results on health-related quality of life and symptom burden. The MANTRA-PAF trial.

AIMS: The Medical ANtiarrhythmic Treatment or Radiofrequency Ablation in Paroxysmal Atrial Fibrillation (MANTRA-PAF) trial assessed the long-term efficacy of an initial strategy of radiofrequency ablation (RFA) vs. antiarrhythmic drug therapy (AAD) as first-line treatment for patients with PAF. In this substudy, we evaluated the effect of these treatment modalities on the Health-Related Quality of Life (HRQoL) and symptom burden of patients at 12 and 24 months. METHODS AND RESULTS: During the study period, 294 patients were enrolled in the MANTRA-PAF trial and randomized to receive AAD (N = 148) or RFA (N = 146). Two generic questionnaires were used to assess the HRQoL [Short Form-36 (SF-36) and EuroQol-five dimensions (EQ-5D)], and the Arrhythmia-Specific questionnaire in Tachycardia and Arrhythmia (ASTA) was used to evaluate the symptoms appearing during the trial. All comparisons were made on an intention-to-treat basis. Both randomization groups showed significant improvements in assessments with both SF-36 and EQ-5D, at 24 months. Patients randomized to RFA showed significantly greater improvement in four physically related scales of the SF-36. The three most frequently reported symptoms were breathlessness during activity, pronounced tiredness, and worry/anxiety. In both groups, there was a significant reduction in ASTA symptom index and in the severity of seven of the eight symptoms over time. CONCLUSION: Both AAD and RFA as first-line treatment resulted in substantial improvement of HRQoL and symptom burden in patients with PAF. Patients randomized to RFA showed greater improvement in physical scales (SF-36) and the EQ-visual analogue scale. CLINICAL TRIAL REGISTRATION: URL http://www.clinicaltrials.gov. Unique identifier: NCT00133211.

Citrulline concentration in routinely collected neonatal dried blood spots cannot be used to predict necrotising enterocolitis.

AIM: Low citrulline concentration is a marker of low functional enterocyte mass, which may predispose neonates to necrotising enterocolitis (NEC). We hypothesised that citrulline could be used to assess the NEC risk that could not be accounted for by gestational age and birthweight. This study investigated whether citrulline concentrations routinely measured in neonatal dried blood spots (DBS) could predict NEC. METHODS: We used national Danish registries to retrospectively identify all 361 babies born between 2003 and 2009 who were diagnosed with NEC and had a valid citrulline concentration measured from a DBS sample. The control group comprised 1083 healthy newborns, with three controls for every newborn with NEC, matched for birthweight and gestational age. RESULTS: Neonatal dried blood spots were collected between 2 and 21 days of life, with a median of 8 days. The results showed that NEC was not associated with low citrulline concentration, either in a direct comparison between the NEC and control groups or in a multivariate logistic regression (p = 0.73). CONCLUSION: The findings of this study show that the citrulline concentrations found in routine DBS samples between 2003 and 2009 did not predict NEC in newborn babies.

The safety of flecainide treatment of atrial fibrillation: long-term incidence of sudden cardiac death and proarrhythmic events.

OBJECTIVE: To assess the safety of long-term treatment with flecainide in patients with atrial fibrillation (AF), particularly with regard to sudden cardiac death (SCD) and proarrhythmic events. DESIGN: Retrospective, observational cohort study. SETTING: Single-centre study at Örebro University Hospital, Sweden. Subjects. A total of 112 patients with paroxysmal (51%) or persistent (49%) AF (mean age 60 ± 11 years) were included after identifying all patients with AF who initiated oral flecainide treatment (mean dose 203 ± 43 mg per day) between 1998 and 2006. Standard exclusion/inclusion criteria for flecainide were used, and flecainide treatment was usually combined with an atrioventricular-blocking agent (89%). MAIN OUTCOME MEASURE: Death was classified as sudden or nonsudden according to standard definitions. Proarrhythmia was defined as cardiac syncope or life-threatening arrhythmia. RESULTS: Eight deaths were reported during a mean follow-up of 3.4 ± 2.4 years. Compared to the general population, the standardized mortality ratios were 1.57 (95% confidence interval (CI) 0.68-3.09) for all-cause mortality and 4.16 (95% CI 1.53-9.06) for death from cardiovascular disease. Three deaths were classified as SCDs. Proarrhythmic events occurred in six patients (two each with wide QRS tachycardia, 1 : 1 conducted atrial flutter and syncope during exercise). CONCLUSION: We found an increased incidence of SCD or proarrhythmic events in this real-world study of flecainide used for the treatment of AF. The findings suggest that further investigation into the safety of flecainide for the treatment of patients with AF is warranted.

Cumulative live birth rates after weight reduction in obese women scheduled for IVF: follow-up of a randomized controlled trial.

STUDY QUESTION: Did weight reduction in obese women scheduled for IVF increase cumulative live birth rate (CLBR) after 2 years? SUMMARY ANSWER: Weight loss prior to IVF did not increase CLBR. WHAT IS KNOWN ALREADY: Few studies have investigated the effect of weight reduction in obese infertile women scheduled for IVF. In a recent randomized controlled trial (RCT), including one IVF cycle, we found no increase in live birth rate after weight reduction. Weight regain after obesity reduction treatment often occurs, and children born to obese women have a higher risk of childhood obesity. STUDY DESIGN SIZE DURATION: A 2-year follow-up of a multicenter, RCT running between 2012 and 2018 was performed. Out of 317 women randomized to weight reduction followed by IVF treatment or IVF treatment-only, 305 remained in the full analysis set. Of these women, 90.5% (276/305) participated in this study. PARTICIPANTS/MATERIALS SETTING METHODS: Nine infertility clinics in Sweden, Denmark and Iceland participated in the RCT. Obese women under 38 years of age having a BMI ≥30 and < 35 kg/m2 were randomized to weight reduction and IVF or IVF-only. In all, 160 patients were randomized to a low calorie diet for 12 weeks and 3-5 weeks of weight stabilization, before IVF and 157 patients to IVF-only. Two years after randomization, the patients filled in a questionnaire regarding current weight, live births and ongoing pregnancies. MAIN RESULTS AND THE ROLE OF CHANCE: 42 additional live births were achieved during the follow-up in the weight reduction and IVF group, and 40 additional live births in the IVF-only group, giving a CLBR, the main outcome of this study, of 57.2% (87/152) and 53.6% (82/153), respectively (P = 0.56; odds ratio (OR) 1.16, 95% CI: 0.74-1.52). Most of the women in the weight reduction and IVF group had regained their pre-study weight after 2 years. The mean weight gain over the 2 years was 8.6 kg, while women in the IVF-only group had a mean weight loss of 1.2 kg. At the 2-year follow-up, the weight standard deviation scores of the children born in the original RCT (index cycle) were 0.218 (1.329) (mean, SD) in the weight reduction and IVF group and - 0.055 (1.271) (mean, SD) in the IVF-only group (P = 0.25; mean difference between groups, 0.327; 95% CI: -0.272 to 0.932). LIMITATIONS REASON FOR CAUTION: All data presented in this follow-up study were self-reported by the participants, which could affect the results. A further limitation is in power for the main outcome. The study is a secondary analysis of a large RCT, where the original power calculation was based on live-birth rate after one cycle and not on CLBR. WIDER IMPLICATIONS OF THE FINDINGS: The follow-up indicates that for women with a BMI ≥30 and < 35 kg/m2 and scheduled for IVF, the weight reduction did not increase their chance of a live birth either in the index cycle or after 2 years. It also shows that even in this highly motivated group, a regain of pre-study weight occurred. STUDY FUNDING/COMPETING INTERESTS: The 2-year follow-up was financed by grants from the Swedish state under the agreement between the Swedish Government and the county councils, the ALF-agreement (ALFGBG-70940 and ALFGBG-77690), Merck AB, Solna, Sweden (an affiliate of Merck KGaA, Darmstadt, Germany), Hjalmar Svensson Foundation. Ms Kluge has nothing to disclose. Dr Bergh has been reimbursed for lectures and other informational activities (Ferring, MSD, Merck, Gedeon Richter). Dr Einarsson has been reimbursed for lectures for Merck and Ferring. Dr Thurin-Kjellberg reports grants from Merck, and reimbursement for lectures from Merck outside the submitted work. Dr Pinborg has been reimbursed for lectures and other informational activities (Ferring, MSD, Merck, Gedeon Richter). Dr Englund has nothing to disclose. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number, NCT01566929.

Cannabis-associated psychosis: Neural substrate and clinical impact.

Prospective epidemiological studies have consistently demonstrated that cannabis use is associated with an increased subsequent risk of both psychotic symptoms and schizophrenia-like psychoses. Early onset of use, daily use of high-potency cannabis, and synthetic cannabinoids carry the greatest risk. The risk-increasing effects are not explained by shared genetic predisposition between schizophrenia and cannabis use. Experimental studies in healthy humans show that cannabis and its active ingredient, delta-9-tetrahydrocannabinol (THC), can produce transient, dose-dependent, psychotic symptoms, as well as an array of psychosis-relevant behavioral, cognitive and psychophysiological effects; the psychotogenic effects can be ameliorated by cannabidiol (CBD). Findings from structural imaging studies in cannabis users have been inconsistent but functional MRI studies have linked the psychotomimetic and cognitive effects of THC to activation in brain regions implicated in psychosis. Human PET studies have shown that acute administration of THC weakly releases dopamine in the striatum but that chronic users are characterised by low striatal dopamine. We are beginning to understand how cannabis use impacts on the endocannabinoid system but there is much still to learn about the biological mechanisms underlying how cannabis increases risk of psychosis. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Body size and prostate cancer: a 20-year follow-up study among 135006 Swedish construction workers.

BACKGROUND: Obesity is associated with endocrine changes (e.g., increased estrogen and decreased testosterone in the blood) that have been implicated in the cause of prostate cancer and, therefore, an association between body weight and the risk of developing prostate cancer would be expected. However, because of bias or low statistical power in previous epidemiologic studies, associations between anthropometric measurements (height and weight), body mass index (BMI), and the risk of prostate cancer may have been inadvertently overlooked. PURPOSE: We performed a large, retrospective cohort study among Swedish construction workers to evaluate possible associations of adult weight, height, BMI, and lean body mass (LBM) by age at entry in the study with the incidence and mortality rate of prostate cancer. METHODS: We analyzed data that had been compiled in a computerized central register on a cohort of approximately 135000 male construction workers. Information on height and weight had been collected with the use of a comprehensive questionnaire filled out by nurses at the time of enrollment in the cohort, from 1971 through 1975. Complete follow-up was achieved through 1991 by means of record linkage to the Swedish National Cancer Register, the Death Register, and the Migration Register. A total of 2368 incident cases and 708 deaths from prostate cancer occurred in the cohort during a follow-up period averaging 18 years. We used only information obtained at the index visit from 1971 through 1975 to determine age-adjusted rate ratios (RRs) in a Poisson-based multiplicative multivariate model with age and the relevant exposure variable (e.g., weight, height, BMI, and LBM) as independent variables. RESULTS: All anthropometric measurements were positively associated with the risk of prostate cancer and were more strongly related to mortality than to incidence. The excess risk of death from prostate cancer was statistically significant in all BMI categories above the reference category: RR = 1.40 (95% confidence interval [CI] = 1.09-1.81) in the highest category compared with the lowest (P for trend = .04). For height and LBM, the excess risk in the highest compared with the lowest categories was somewhat less pronounced: RR = 1.28 (95% CI = 1.02-1.60) and RR = 1.26 (95% CI = 1.02-1.57), respectively. Statistically significant linear dose-response relationships were also found with the incidence of prostate cancer, with the exception of BMI (P for trend = .10). CONCLUSION: Our large cohort study indicates that various aspects of body size are related to the risk of prostate cancer and that future studies are needed to study the role of body size and prostate cancer.

The isoelectric point of the human red cell glucose transporter.

The isoelectric point (pI) of the human red cell glucose transporter (Glut 1) was determined. Inconsistent values of 6.0, 6.4-6.5 and 8 have been reported earlier. Integral membrane proteins from human red cells were analyzed by two-dimensional electrophoresis with isoelectric focusing and sodium dodecyl sulfate gel electrophoresis (2D-PAGE). A zone of monomeric Glut 1 was found at pH 8.7, but most of the Glut 1 focused at pH 6-7 together with the anion transporter and other components. Purified Glut 1 focused only at pH 8.5 +/- 0.2 (S.D., n = 12) and deglycosylated purified Glut 1 only at pH 8.4 +/- 0.1 (n = 5), as shown by 2D-PAGE. The absence of Glut 1 below pH 8 in the latter cases was confirmed by immunoblotting with a monoclonal antibody. Furthermore, Glut 1 was photoaffinity-labelled with [3H]cytochalasin B and subjected to isoelectric focusing in one dimension. The pI of the labelled Glut 1 was 8.6 +/- 0.3 (n = 11). A pI of 9.1 was calculated for the Glut 1 polypeptide on the basis of amino acid composition and pKa values for amino acid side groups. The sialic acid content of the glycosylated transporter from fresh red cells was determined at approximately 2.1 sialic acid residues per transporter, which corresponds to a calculated pI of 8.8. The pI values of other human glucose transporter polypeptides of the facilitative diffusion type (Glut 2, 3, 4 and 5) were calculated at 8.4, 7.4, 7.1 and 6.2, respectively.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine.

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the Mr of the Glut 1 polypeptide was estimated to be 49,000 +/- 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 +/- 0.03 ml mg-1 cm-1 at 280 nm, referring to the polypeptide concentration. The Mr value is consistent with the cDNA-deduced Mr 54,117 of the very similar HepG2 glucose transporter polypeptide. At 2 degrees C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

Monomeric human red cell glucose transporter (Glut1) in non-ionic detergent solution and a semi-elliptical torus model for detergent binding to membrane proteins.

The self-association state of the human red cell glucose transporter (Glut1) in octaethylene glycol n-dodecyl ether (C12E8) and n-octyl beta-D-glucopyranoside (OG) solution was analyzed in the presence of reductant by gel filtration with light-scattering, refractivity and absorbance detection, and by ultracentrifugation. The C12E8-Glut1 complex was essentially monomeric, whereas OG-Glut1 also formed dimers and larger oligomers. C12E8-Glut1 retained substantial glucose transport activity even after depletion of endogenous lipids by gel filtration, as shown by reconstitution and transport measurements. Removal of endogenous lipids from OG-Glut1 abolished the activity unless phosphatidylcholine was included in the eluent. The binding of C12E8 and OG to Glut1 was determined by gel filtration with refractivity and absorbance detection or with radioactive tracer to be 1.86 +/- 0.07 and 1.84 +/- 0.09 g/g polypeptide, respectively. A structural model was proposed in which non-ionic detergent forms a semi-elliptical torus (SET) surrounding the transmembrane protein. The torus thickness was assumed to be equal to the radius (short half-axis) of a spherical (oblate ellipsoidal) free detergent micelle and the polar head groups of the detergent molecules were predicted to be situated just outside the hydrophobic surface of the protein. The experimental detergent binding values and those obtained from the SET model together confirmed that Glut1 was monomeric in C12E8 solution and provided constraints on the shape and size of the hydrophobic transmembrane region of Glut1 in alpha-helical and beta-barrel topology models.

Circadian variation of malignant ventricular arrhythmias in patients with ischemic and nonischemic heart disease after cardioverter defibrillator implantation. European 7219 Jewel Investigators.

OBJECTIVES: The purpose of this study was to examine the circadian variation of ventricular arrhythmias detected by an implantable cardioverter defibrillator in patients with and without ischemic heart disease. BACKGROUND: Previous studies have shown a circadian variation of ventricular arrhythmias, sudden death and myocardial infarction with a peak occurrence in the morning hours. The circadian pattern, which is similar for both arrhythmic and ischemic events, suggests that ischemia may play a critical role in the genesis of ventricular arrhythmias and sudden death. We hypothesized that, if ischemia plays an important role in the triggering of ventricular arrhythmias, the circadian pattern should be different in patients with ischemic heart disease compared with patients with nonischemic heart disease. METHODS: The circadian variation of ventricular arrhythmias recorded by an implantable cardioverter defibrillator was studied in 310 patients during a mean follow-up of 181 +/- 163 days. Two hundred four patients had a history of ischemic heart disease and 106 patients had nonischemic heart disease. The times of the episodes of ventricular arrhythmias were retrieved from the data log of each device during follow-up, and the circadian pattern was compared between the two groups. RESULTS: During follow-up, 1,061 episodes of ventricular arrhythmias were recorded by the device in the 310 patients. Six hundred eighty-two episodes occurred in the group of patients with ischemic heart disease and 379 occurred in the nonischemic heart disease group. The circadian variation of the episodes showed a typical pattern with a morning and afternoon peak in both groups of patients with ischemic and nonischemic heart disease, but there was no significant difference between the two groups. CONCLUSIONS: The circadian rhythm of ventricular arrhythmias in patients with ischemic heart disease is similar to patients with nonischemic heart disease, suggesting that the trigger mechanisms of the initiation of ventricular tachyarrhythmias may be similar, irrespective of the underlying heart disease.

Diagnostic value of programmed ventricular stimulation in patients with bifascicular block: a prospective study of patients with and without syncope.

OBJECTIVES: The aim of this study was to examine the inducibility of ventricular arrhythmias in patients with bifascicular block both with and without a history of syncope and to relate the findings to clinical events during follow-up. BACKGROUND: Patients with bifascicular block have an increased risk of sudden death that is not reduced by pacemaker treatment. This risk could be related to a high incidence of ventricular arrhythmias. METHOD: Programmed ventricular stimulation was performed in 101 patients with bifascicular block: 41 had a history of unexplained syncope, and 60 were asymptomatic. RESULTS: Programmed ventricular stimulation resulted in a sustained ventricular arrhythmia in 18 patients (18%), 8 in the syncope group and 10 in the nonsyncope group (p = NS). Three patients in each group had an inducible sustained monomorphic ventricular tachycardia. During a mean follow-up of 21 months, 10 patients experienced a clinical event defined as sudden death (n = 4), syncope (n = 5) or appropriate discharges from an implantable cardioverter-defibrillator (n = 1). Only one of these patients had an inducible ventricular arrhythmia at baseline. CONCLUSIONS: The inducibility of ventricular arrhythmias is high in patients with bifascicular block and of the same magnitude in patients with and without a history of syncope. Clinical events during follow-up were not predicted by programmed ventricular stimulation in either of the two groups. The finding of inducible ventricular arrhythmia in patients with bifascicular block should therefore be interpreted with caution.

Recent achievements and research initiated in the Swedish plastics and rubber industry.

The improvement in exposure conditions in the Swedish vinyl chloride producing industry is reported. The article comments on the technology and control methods by which the vinyl chloride concentration has been lowered to less than 1 ppm vinyl chloride. Two epidemiological retrospective cohort studies are presently under way on workers in PVC-utilizing industries and in the rubber industry.

Mortality and cancer morbidity in a group of Swedish VCM and PCV production workers.

The cohort of workers employed in a Swedish vinyl chloride/poly(vinyl chloride) plant since its start in the early 1940's has been followed for mortality and cancer morbidity patterns. Only 21 of the 771 persons could not be traced. Difficulties in establishing exposure levels at different work areas in the past makes an evaluation of dose-effect relationships impossible. A four- to fivefold excess of pancreas/liver tumors was found, including two cases later classified as angiosarcomas of the liver. The number of brain tumors and suicide do not deviate significantly from expected. Cardiovascular and cerebrovascular diseases, on the other hand, differ significantly from the expected. The discrepancies between previous reports on VCM/PVC workers and this report are discussed. The possible etiology of the cardiovascular deaths is also discussed.