Single-cell transcriptome analysis of endometrial tissue.

STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.

Glucose evaluation trial for remission (GETREM) in type 1 diabetes: a European multicentre study.

OBJECTIVE: Strict metabolic control during the 1st year of type 1 diabetes is thought to be a key factor for achieving clinical remission. The aims of this study were two-fold: (i) to evaluate the frequency and duration of spontaneous remission (defined according to the parameters issued by the International Diabetic Immunotherapy Group (IDIG)) in a European population of consecutive recent onset type 1 diabetes patients (aged 5-35 years), followed-up for a period of 36 months with a common protocol of intensive insulin therapy and without adjunct immune-intervention; and (ii) to identify the predictive factors for clinical remission. RESEARCH DESIGN AND METHOD: A total of 189 consecutive patients with newly diagnosed type 1 diabetes according to ADA criteria were recruited in participating centres (Belgium, Czech Republic, Estonia, France, Germany, Hungary, Italy, Poland, Romania, Sweden and Turkey) and followed-up for a period of up to 36 months. In all patients, intensive insulin therapy was implemented consisting of three or four injections of regular insulin daily with NPH insulin at bedtime. Adjustment of insulin dose was made according to a common protocol. Various clinical characteristics (age, gender, severity of presentation, etc.), history (presence of diabetic siblings in the family, etc.) and integrated parameters of metabolic control (HbA(1c), blood glucose, the total insulin dose at hospital discharge adjusted for body weight) were collected. RESULTS: Twenty-two patients (11.6%) experienced remission. The median duration of remission was 9.6 months and the range was 31 months. There was a wide variation among centres. Logistic regression analysis focused on the centre as the main variable in achieving remission. CONCLUSION: Remission was shown to be very heterogeneous between centres depending on 'other factors' such as patient care and family awareness of the disease rather than on 'measurable factors' such as sex, age, HbA(1c) and severity of presentation at diagnosis. Using intensive insulin therapy and optimisation of metabolic control, remission occurred in nearly one out of eight patients.

Patient with type 2 diabetes and stroke.

A case is presented of a type 2 diabetic patient who had a stroke. Various treatment issues are discussed.

Increasing incidence of childhood-onset type I diabetes in 3 Baltic countries and Finland 1983-1998.

AIMS/HYPOTHESIS: We aimed to study the incidence of Type I diabetes in 4 countries, Estonia, Latvia, Lithuania and Finland, during 1983-1998, focusing on the two separate periods of 1983-1990 and 1991-1998. METHODS: Population-based incidence data from nationwide diabetes registries were used. Crude and age-standardized incidence rates using the proportions of 39%, 32% and 29% for 5-year age groups (0-4, 5-9 and 10-14 years) were calculated. Yearly incidence was evaluated and the means between the two periods compared. RESULTS: Between 1983-1990 and 1991-1998 there was a statistically significant incidence increase in all 4 countries of Estonia, Lativia, Lithuania and Finland (relative risk 1.15, 95%-Confidence interval 1.10-1.19) and as well as in the 3 Baltic states of Estonia, Latvia, Lithuania (relative risk 1.13, 95%. Confidence interval 1.04-1.22). The crude incidence increased in Estonia from 10.1 (95%-Confidence interval 8.9-11.4) to 12.3 (11.0-13.8), in Latvia from 6.6 (5.8-7.3) to 7.4 (6.6-8.2) and in Lithuania from 6.8 (6.2-7.5) to 7.8 (7.1-8.5). In Finland the incidence rose from 34.6 (33.3-36.0) in 1983-1990 to 40.8 (39.4-42.2) in 1991-1998. In children under 5 years of a age a statistically important increase was seen in Estonia and Finland. The highest incidence for a single year was recorded for all participating countries in the late 1990 s. The highest annual incidence rate of childhood onset Type I diabetes in the world ever known was recorded in Finland in 1998 with 48.5 cases per 100 000 person-years. CONCLUSION/HYPOTHESIS: The incidence of Type I diabetes has increased since 1983 in the three Baltic states as well as in Finland. Long-term monitoring is needed for a better detection in changes in incidence.

Diabetes and social conditions in Estonia. A population-based study.

BACKGROUND: The aim of this study was to investigate aspects of metabolic control, treatment and complications as well as quality of life in patients with diabetes mellitus from a defined population in Estonia. METHODS: We invited 220 randomly selected diabetes patients recruited to a clinical investigation from a local diabetes register of 1,100 patients in Viljandi, Estonia. The main outcome measures were derived from medical history, physical examination (height, weight and blood pressure), laboratory variables (blood glucose and glycated haemoglobin A1 (HbA1 normal reference range 3.2-5.6%), serum total and HDL cholesterol and creatinine), a questionnaire on disease-related knowledge and quality of life variables. RESULTS: In all, 181 diabetes patients were investigated, of whom 90% were diagnosed with type 2 diabetes. The mean diabetes duration was 8.9 years from clinical diagnosis and mean HbA1 level was 7.3%. The overall proportion of patients treated with insulin was 29.8% and with anti-hypertensive drugs 26.5%. Smoking was present in 14.3%. The proportion of patients with various diabetes complications was high (73.5%), mostly consisting of different manifestations of cardiovascular disease. Foot ulcers or gangrene were observed in 11.6%. A low level of quality of life was registered in many patients, mostly due to difficult living conditions. CONCLUSIONS: Diabetes patients in Viljandi showed an acceptable degree of glucose metabolic control, but reported a high degree of diabetes complications, as well as impaired quality of life. The diabetes complications may therefore be due to detrimental factors other than hyperglycaemia, e.g. the standard of care during previous years as well as current social and living conditions.

Mortality in patients with childhood-onset type 1 diabetes in Finland, Estonia, and Lithuania: follow-up of nationwide cohorts.

OBJECTIVE: To assess mortality of population-based cohorts of childhood-onset type 1 diabetic patients from the Eastern European countries of Estonia and Lithuania and compare this information with recent data from Finland. RESEARCH DESIGN AND METHODS: Estonian (n = 518) and Finnish (n = 5,156) type 1 diabetic cohorts were diagnosed between 1980 and 1994, and the Lithuanian (n = 698) cohort was diagnosed between 1983 and 1994. The mortality of these cohorts was determined in 1995. Life-table analysis, Cox survival analysis with covariates, and standardized mortality ratios (SMRs) were used. Causes of death were analyzed. RESULTS: Survival after 10 years duration of type 1 diabetes was similar in Estonia (94.3%) and Lithuania (94.0%), but much higher in Finland (99.1%). In the Cox survival analysis with covariates, the country of origin and age at diagnosis were found to be significant predictors of mortality. The SMR for the Estonian cohort was 4.35 (95% CI 2.25-7.61), the highest for the Lithuanian cohort was 7.55 (4.89-11.15), and the lowest for the Finnish cohort was 1.62 (1.10-2.28). The most common cause of death in Estonia and Lithuania was diabetic ketoacidosis (DKA), and in Finland, it was violent causes. No deaths from late complications of diabetes have been documented so far in any of the three countries. CONCLUSIONS: Our results demonstrate a high rate of short-term deaths due to DKA and inferior survival of childhood-onset type 1 diabetic patients in Estonia and Lithuania compared with Finland. In Finland, the survival of childhood-onset type 1 diabetic patients has improved and is only slightly inferior to that of the background population.

Seasonal variation in the incidence of Type 1 diabetes mellitus during 1983 to 1992 in the countries around the Baltic Sea.

AIM: To examine seasonal patterns of incidence of Type 1 diabetes mellitus incidence in children aged 0-14 years in Finland, Sweden, Estonia, Latvia and Lithuania during 1983-1992 (1987-1992 for Finland). METHODS: The study used a method that models incidence data using combinations of sine waves to model seasonal variation around a possible linear trend. RESULTS: In Finland, a significant pattern was found for combined sexes and age groups 0-9 and 10-14 years. A significant pattern was also confirmed for 10-14 year-old boys. In Sweden, the best model with significant pattern was found separately for boys and girls and age groups 0-9 and 10-14 years, however, a significant pattern was confirmed for older girls only. A seasonal pattern in older boys in Finland and girls in Sweden was characterized by two cycles with decreased incidence in June and November-December. The pattern among younger children (0-9 or 5-9 years) had one cycle with a decreased incidence in May-June. In Estonia, a significant pattern was found for the age group 0-14 years and combined sexes. No significant seasonal patterns were found in Latvia and Lithuania. CONCLUSIONS: The seasonal pattern with two cycles among older children and one cycle only among younger children may indicate different triggers of Type 1 diabetes mellitus for different age groups.

Glutamic acid decarboxylase and islet cell antibodies in healthy Estonian children.

The prevalence of antibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) was compared with that of islet cell antibodies (ICA) in 614 non-diabetic Estonian children (314 males) aged 3-18 years representing the general population. GADA were analyzed with a radioligand assay, and ICA with a standard immunofluorescence method with a detection limit of 2.5 Juvenile Diabetes Foundation (JDF) units. Fourteen subjects (2.3%, 95% confidence interval [CI] 1.1-3.5%) tested positive for GADA (median level 10.8 relative units [RU], range 7.7-154.2 RU), while 10 (1.6%, CI 0.6-2.6%) had ICA (median levels 34 JDF units, range 3-97 JDF units). Five subjects (0.8%, CI 0.1-1.5%; p = 0.03 vs GADA and 0.15 vs ICA) were double positive. The individual with the second highest GADA level (129.3 RU) and the highest ICA level (97 JDF units) presented with type 1 diabetes 4 months later. A follow-up sample was obtained approximately 3-4 years after the first sampling in 14 subjects initially positive for ICA and/or GADA. Four of the nine initially ICA-positive children remained positive, but their levels decreased from a median of 42 to 18 JDF units (p = 0.06). Only two of the nine retested subjects initially positive for GADA remained positive in the second sample. These observations suggest that the prevalence of GADA in non-diabetic children is of the same magnitude as that of ICA. Combined positivity for both GADA and ICA is less prevalent than single antibody specificities, indicating that double autoantibody positivity may have a higher predictive value for future type 1 diabetes in the general population than either antibody separately. The evanescent character of diabetes-associated autoantibodies in a proportion of the unaffected children implies that more subjects may experience self-restricted beta-cell damage than the number progressing to actual disease.

Distribution of insulin-dependent diabetes mellitus (IDDM)-related HLA alleles correlates with the difference in IDDM incidence in four populations of the Eastern Baltic region.

The high incidence of insulin-dependent diabetes mellitus (IDDM) in Finland contrasts strikingly with the low rates in the neighbouring populations of countries in the Eastern Baltic region: Estonia, Latvia and Russia. To evaluate the possible contribution of genetic factors to these differences, the frequencies of HLA-DQB1 alleles and relevant DQB1-DQA1 or DQB1-DRB1 haplotypes associated with IDDM risk or protection were analysed among IDDM patients and control subjects from these four populations. An increased frequency of HLA-DQB1*0302, DQB1*02-DQA1*05 and DQB1*0302-DRB1*0401 was observed in subjects with IDDM in all studied populations, whereas the prevalence of DQB1*0301 and DQB1*0602 and/or *0603 was decreased among patients. The degree of IDDM risk associated with HLA alleles analysed here did not differ significantly between the populations. Comparisons of the distribution of IDDM-related HLA alleles and haplotypes in the background populations revealed its consonance with IDDM incidence. The combined frequency of high risk genotypes was significantly higher among Finns than in other populations studied. Our data support the hypothesis that variance in the dispersion of HLA alleles is the genetic basis of variation of IDDM incidence observed in the Eastern Baltic region.

Effects of citalopram treatment on behavioural, cardiovascular and neuroendocrine response to cholecystokinin tetrapeptide challenge in patients with panic disorder.

Eight patients with panic disorder were administered 20 micrograms of cholecystokinin tetrapeptide (CCK-4) before and after 8 weeks of treatment with the selective serotonin reuptake inhibitor (SSRI) citalopram. All patients responded to treatment by showing a significant general improvement and reaching a panic-free state for 2 weeks. At the rechallenge with CCK-4, patients displayed a marked reduction in the intensity and number of panic symptoms. The frequency of panic attacks induced with CCK-4 decreased by 50% after treatment. Citalopram treatment had no substantial effect on cardiovascular (heart rate and blood pressure) or hormonal (cortisol, prolactin and growth hormone) responses to CCK-4. Patients who still had panic attacks after treatment demonstrated a blunted growth hormone response to CCK-4 that was not seen in those who did not have panic attacks. This study suggests that treatment with an SSRI can reduce an enhanced sensitivity to CCK-4 without modifying cardiovascular and neuroendocrine responses to CCK-4 in patients with panic disorder.

Incidence trends in childhood onset IDDM in four countries around the Baltic sea during 1983-1992.

We present secular trends of childhood onset insulin-dependent diabetes mellitus (IDDM) in Finland, Estonia, Latvia and Lithuania during the period of 1983-1992. Incidence data were obtained from the national IDDM registries. The average age-standardized incidence per 100,000/year was 35.0 in Finland, followed by 10.2 in Estonia, 7.1 in Lithuania and 6.5 in Latvia. A male excess in incidence was recorded in Finland (1.15) and Latvia (1.01). In all countries, the highest age-specific risk of IDDM was observed in the 11-13 year age range. The large difference in incidence between Finland and other Baltic countries was see even in 1-2 year-old children. During the 10-year study period overall changes in incidence of IDDM were relatively small in these four countries. The incidence increased in Finland and Lithuania on average by 1% and 1.4% per year, respectively. A statistically significant increase was recorded only in 0-4 year old children in Finland, at 5.6% per year. In Estonia, an 8.3% increase in this age group, however, was not statistically significant. The different trends in the age-group specific incidence rates were confirmed in Finland. In conclusion, from 1983 to 1992 the incidence of childhood onset IDDM was increasingly in Finland and Lithuania, while in Latvia and Estonia it was stable. There are still great differences in IDDM incidence between the countries around the Baltic Sea.

No deterioration of glucose tolerance in weight cycling obese.

OBJECTIVE: To evaluate whether weight cycling is detrimental to the changes in glucose tolerance in obese individuals without overt NIDDM and related to the amplitude of weight cycling. DESIGN: Historical prospective observational study of a hospital-based cohort. SUBJECTS: One hundred twenty-five obese individuals drawn from the medical records of the Hospital of Endocrinology, University of Tartu, in whom at least one weight cycle was detected. Selected cutoff value for weight cycling set to 3, 6, 9 and 12 kg of weight loss and subsequent regain. MEASUREMENTS: Weight measurements and oral glucose tolerance tests. The latest oral glucose tolerance test and the one during the first visit compared by the 2 h blood glucose values and areas under the blood glucose curve. RESULTS: No deterioration of glucose tolerance recorded in any of the groups with different cutoff values for weight cycling. No trend towards the deterioration of glucose tolerance with increasing amplitude of weight cycles. CONCLUSION: We cannot claim that weight cycling is detrimental to glucose tolerance in non-diabetic obese individuals. This effect is independent of the amplitude of weight cycling. Weight reduction may be recommended to obese individuals for the prevention of NIDDM even if it is unsuccessful and the phenomenon of weight cycling results.

Evidence for importance of gender and birth cohort for risk of IDDM in offspring of IDDM parents.

The risk of developing diabetes is higher in offspring of fathers than of mothers with insulin-dependent diabetes mellitus (IDDM). The reasons for this sex differential are unclear, as early studies were often selected and relatively small. We conducted a prospective study on the risk of IDDM in a cohort of 9,453 offspring from 5,255 Finnish parents with diabetes diagnosed before age 30 years. Age of first admission to the hospital was considered to be the age of diagnosis of IDDM in the offspring; IDDM occurred in 248 offspring. The risk of IDDM tended to be lower in the offspring of the same gender as the diabetic parent (adjusted risk ratio (RR) 0.78; p = 0.50). When offspring were of same gender as the diabetic parent, male offspring had a higher risk of IDDM than female offspring (RR 2.28; 95% confidence interval 1.53-3.38), whereas if the gender of the diabetic parent and the offspring were different, the risk in male offspring was lower (RR 0.43; 95% confidence interval 0.31-0.62). For the offspring of diabetic fathers, the cumulative risk by the age of 20 was higher (7.6%) than for those with diabetic mothers (3.5%) (p < 0.0001). In a multivariate analysis statistically significant predictors of IDDM in the offspring were the sex of the parent, the year of birth and the birth order of the offspring. The risk of IDDM in the offspring increased by 9% per year of birth cohort. By age 20, the cumulative risk of developing IDDM in the offspring of diabetic parents was 5.3%, 10 times higher than in the background population. It is likely that genetic factors seem to have played a major role in the continuous increase of IDDM incidence in Finnish children.

Incidence of childhood diabetes did not increase in Estonia during 1980-89.

The purpose of the study was to evaluate the temporal variation in the incidence of Type 1 diabetes in Estonia for 1980-89. Data were taken from the Estonian Childhood Type 1 Diabetes Register. "Poisson" regression modelling was completed with GLIM software adjusting for age and ethnic groups. No calendar period effect was found, either when allowing for distinct period effects, or looking for trends. No urban-rural difference was detected in the incidence of childhood onset Type 1 diabetes. In conclusion, our analysis provides evidence that the incidence of childhood Type 1 diabetes was relatively stable in Estonia during the past decade in contrast to some other Baltic countries where a significant increase has been reported.

Risk of childhood type 1 diabetes for Russians in Estonia and Siberia.

We have compared the incidence of Type 1 diabetes in childhood between two predominantly Russian populations from the former Soviet Union--the non-Estonians living in Estonia and the inhabitants of the district of Novosibirsk. The study period covered the years 1980-1989 for the non-Estonians and 1983-1989 for the district of Novosibirsk. The mean annual incidence of Type 1 diabetes was significantly higher in the non-Estonian population, 7.8 per 100,000 (95% confidence interval (CI): 6.3-9.6) than in Novosibirsk where it was 4.7 per 100,000 (95% CI: 4.1-5.4). The highest incidence in females from Estonia was in the age group 5-9 years and in Novosibirsk 10-14 years. In the youngest age group of 0-4 years there was no difference in the incidence between non-Estonians in Estonia and the population of Novosibirsk, or males and females in either population. There was no difference in the incidence between the 0-4 and 5-9 year age groups in Novosibirsk males. No time trend was seen in the incidence over the study period in either population. The annual incidence in Novosibirsk was fairly stable, while in the non-Estonians it showed two distinct peaks. The most likely reason for the observed phenomena is a different pattern and higher prevalence of environmental causal agents in the Baltic country of Estonia.

Low incidence of childhood IDDM in district of Novosibirsk (Russia).

OBJECTIVE: To study the incidence of insulin-dependent diabetes mellitus (IDDM) among 0- to 14-yr-old children of the district of Novosibirsk, in western Siberia, Russia, from 1983 to 1989. RESEARCH DESIGN AND METHODS: Data were collected with methods recommended by the Diabetes Epidemiology Research International Group. RESULTS: The average yearly incidence among males and females was 4.7 (95% confidence interval 3.9-5.8) and 4.4 (95% confidence interval 3.6-5.4) per 100,000 people, respectively. The incidence of IDDM among children living in the city of Novosibirsk was significantly higher than in children living in the surrounding counties of the district, 5.7/100,000 (95% confidence interval 4.8-6.8) versus 3.6/100,000 (95% confidence interval 2.9-4.5). CONCLUSIONS: The incidence of childhood IDDM among Russians in Siberia is low. This is the first publication of internationally comparable data on the occurrence of childhood IDDM in the Russian population.

Comparison of the incidence of insulin-dependent diabetes mellitus in childhood among five Baltic populations during 1983-1988.

We have carried out a comparison of the incidence of childhood onset insulin-dependent diabetes mellitus (IDDM) between five populations around the Baltic Sea. These were Finland, Estonia, Latvia, Lithuania and Poland. The risk of IDDM is highest in the world in Finland and also very high in Sweden, on the western and northern side of the Baltic Sea. The risk of IDDM in children on the eastern side of the Baltic Sea has not been known before. The data collection period covered the years 1983-1988. A marked variation in incidence was seen within this relatively small geographical area. Among these five populations, the incidence increased with the latitude. Our present results confirmed the very high incidence of IDDM in Finland. The average age-standardized yearly incidence of IDDM/100,000 was in males under 15 years of age 36.9 in Finland, 10.7 in Estonia, 6.4 in Latvia, 6.5 in Lithuania and 6.0 in Poland. In females the incidence was 31.6, 10.0, 6.9, 7.0 and 6.4 in these five populations, respectively. The differential in incidence in Estonia as compared with Latvia, Lithuania and Poland was statistically significant (P = 0.0002). A slight male excess in incidence was found in countries with higher incidence--Finland and Estonia, but in lower incidence countries the sex ratio was opposite (P = 0.019 for the interaction sex-population). During 1983-1988 the incidence increased significantly in Finland but not in other populations although a large year-to-year variation in incidence was observed in each country, particularly in males. We recorded a peak in IDDM incidence in most of these populations around 1986.