Pekka J. Karhunen

Background: This study characterizes microbial relationships and bacterial transfer across the rectal, vaginal and endometrial microbiomes upon embryo transfer, and evaluates the influence of obesity on site-specific profiles. The Tampere Infertility Microbiome Project (TIMP) included eighty-nine women with male-factor infertility undergoing IVF or ICSI. Participants were classified into normal-weight ( n = 45, BMI < 25 kg/m ² ) and obese ( n = 44, BMI 30–35 kg/m ² ) groups. A total of 267 rectal, vaginal and endometrial samples were collected upon fresh embryo transfer and analysed using next-generation sequencing. Results: The anatomical site was the dominant determinant of microbial structure. Rectal samples showed the highest diversity and differed from Lactobacillus -dominated vaginal and endometrial communities ( p < 0.0001), which partially overlapped but exhibited distinct genera. Lactobacillus accounted for 61.3% of vaginal, 18.2% of endometrial and 1.7% of rectal communities. Higher vaginal abundance of Lactobacillus was associated with lower occurrences of Gardnerella , Prevotella and other non- Lactobacillus genera ( p = 0.001), suggesting a vaginal-endometrial association. Increased rectal abundances of Gardnerella and Prevotella corresponded to higher vaginal abundances of the same genera, suggesting a rectal-vaginal microbial relationship. Obesity was associated with a trend towards increased vaginal diversity and a higher prevalence of Gardnerella in obese versus normal-weight individuals (19% vs. 9%, p = 0.047). Conclusions: Rectal microbiomes were diverse and distinct from vaginal and endometrial communities. Vaginal Lactobacillus dominance was associated with lower abundance of potentially pathogenic genera. Microbial continuity was most pronounced for Gardnerella and Prevotella . BMI-related variation appeared most pronounced in the vaginal microbiome, followed by the endometrium, whereas community-level differences between BMI groups were not statistically significant.

Background: Bacterial DNA from the oral cavity, respiratory tract, gut, and skin has been detected in atherosclerotic plaques, suggesting a role in chronic inflammation linked to atherosclerosis. Chronic bacterial infections often form biofilms resistant to antibiotics and immune detection, giving rise to a new generation of virulent bacteria in suitable conditions. This study explores the role of the immune system in bacterial‐induced inflammation of atherosclerotic plaques. Methods: Coronary plaques from 121 sudden death victims and endarterectomy samples from 96 surgical patients were analyzed using bacterial real‐time quantitative polymerase chain reaction, immunohistochemistry, and genome‐wide expression analysis. TLR (toll‐like receptor) signaling was examined in bacterial‐activated TLR cell lines. Results: Of the bacteria detected, oral viridans group streptococcal DNA was the most common, being found in 42.1% of coronary plaques and 42.9% of endarterectomies. Immunopositivity for viridans streptococci correlated with severe atherosclerosis ( P P =0.021) or myocardial infarction ( P =0.042). Viridans streptococci colonized the core of the atheroma as a biofilm unrecognized by macrophages of the innate immune system. In contrast, immunopositive streptococci that appeared to have originated from the biofilm infiltrated the ruptured fibrous cap of the atheroma in endarterectomy samples and coronary plaques and were detected by pattern‐recognizing receptors and coexpressed with the adaptive immune response. Among the viridans streptococcal strains, TLR2 was the most activated bacterial‐signaling pathway. Genome‐wide expression analysis of endarterectomy samples showed upregulation of bacterial recognition pathways. Conclusions: Latent chronic bacterial inflammation evades immune detection and may contribute to the pathogenesis of complicated atherosclerotic plaques and fatal myocardial infarction.

Cigarette smoking is the single most significant cause of preventable death in the world. Tobacco smoking causes exposure to thousands of chemicals and disrupts biological pathways. It impacts several organs, including the brain, where its effects are mediated by nicotinic acetylcholine receptors. Women seem to be more susceptible to the negative health effects of smoking. In this study, we focused on the changes in the metabolic profile of human postmortem frontal cortex and cerebrospinal fluid (CSF) samples associated with high levels of the nicotine metabolite cotinine. We used non‐targeted metabolomics to analyse post‐mortem frontal cortex and CSF samples from the Tampere Sudden Death Study cohort. We identified 137 cases (24 females) with high cotinine levels, indicating nicotine exposure. For controls, we identified 82 subjects (20 females) with no cotinine in the frontal cortex or CSF samples and no known history of smoking based on medical records and autopsy reports. Cases had significantly higher levels of 1‐methylhistamine (Cohen's d = 0.66, p < 0.0001) and N‐acetylputrescine (d = 0.84, p < 0.0001), and lower levels of aspartic acid (d = −0.53, p < 0.001), 3‐methylhistidine (d = −0.58, p = 0.0004) and taurine (d = −0.47, p = 0.0002) in the frontal cortex compared to controls. Compared to the frontal cortex, differences between cases and controls were smaller in the CSF samples. Most of the observed differences were similar in both sexes, with a few exceptions like low ergothioneine levels, observed especially in female cases. In conclusion, smoking or nicotine exposure is associated with alterations in metabolites linked to increased oxidative stress and neuroinflammation, as well as reduced neurotransmitter levels in the frontal cortex.

Background: Atrophic gastritis—the end stage of chronic gastritis—is an asymptomatic disease due to Helicobacter pylori infection causing decreased vitamin B12 and folate absorption, which may lead to severe haematological and neuropsychological disorders including Alzheimer’s disease. The diagnosis requires endoscopy and biopsies from symptomatic patients, explaining why its true prevalence in the population is not well-known. Objective: We aimed to evaluate the prevalence of various stages of chronic gastritis in an autopsy series most closely representing the general population. Subjects and Methods: Gastric mucosa samples were collected prospectively from out-of-hospital deaths included in the Tampere Sudden Death Study (n = 70, mean age 63, age range 22–91 years). Antrum and corpus samples were stained with a H. pylori antibody and staged histopathologically. Results: Chronic gastritis with or without atrophic changes was detected in 40% of the cases. The proportion of healthy mucosa decreased age-dependently from 71.4% among individuals aged 70 years), and that of chronic non-atrophic gastritis from 21.4% to 8.7%. In contrast, the prevalence of atrophic gastritis was 27.1% and increased in the age groups from 7.1% to 47.8% (P = .019) among the oldest individuals, showing a strong association (P < .0001) with H. pylori immunopositivity. Conclusions: Atrophic gastritis is a common feature of the ageing stomach, which is observed in every second individual aged 70+ years, showing a strong association with H. pylori immunopositivity. Atrophic gastritis may be a more common risk factor in old age for diseases associated with low serum B12 and folate levels than has been previously known.

Cigarette smoking is the single most significant cause of preventable death in the world. Tobacco smoking causes exposure to thousands of chemicals and disrupts biological pathways. It impacts several organs, including the brain, where its effects are mediated by nicotinic acetylcholine receptors. Women seem to be more susceptible to the negative health effects of smoking. In this study we focused on the changes in the metabolic profile of human postmortem frontal cortex and cerebrospinal fluid samples associated with high levels of the nicotine metabolite cotinine. We used non-targeted metabolomics to analyze post-mortem frontal cortex and cerebrospinal fluid (CSF) samples from the Tampere Sudden Death Study cohort. We identified 137 cases (24 females) with high cotinine levels, indicating nicotine exposure. For controls, we identified 82 subjects (20 females) with no cotinine in the frontal cortex or CSF samples and no known history of smoking based on medical records and autopsy reports. Cases had significantly higher levels of 1-methylhistamine (Cohen’s d=0.66, p<0.0001) and N-acetylputrescine (d=0.84, p<0.0001), and lower levels of aspartic acid (d=-0.53, p<0.001), 3-methylhistidine (d=-0.58, p=0.0004), and taurine (d=-0.47, p=0.0002) in the frontal cortex compared to controls. Compared to the frontal cortex, differences between cases and controls were smaller in the CSF samples. Most of the observed differences were similar in both sexes, with a few exceptions like low ergothioneine levels, observed especially in female cases. In conclusion, smoking or nicotine exposure is associated with alterations in metabolites linked to increased oxidative stress and neuroinflammation, as well as reduced neurotransmitter levels in the frontal cortex.

Background: Molecular microbial techniques have identified several DNA sequences from oral and gut bacteria in atherosclerotic plaques. The composition of the plaque microbiome has shown great heterogeneity due to varying DNA extraction and amplification methods, small size sample cohorts, and a low abundance of bacteria in plaques, leading to interference by contaminating bacterial sequences from laboratory chemicals. We used a novel next-generation sequencing (NGS) approach to analyze the entirety of bacterial DNAs found in coronary artery plaques in a unique prospective autopsy series of out-of-hospital deaths representing a cross-section of the general population. Methods: DNA was extracted from aseptically collected and frozen (−80 ^° C) atherosclerotic coronary plaque samples taken from the left anterior descending (LAD) coronary arteries of 202 victims of sudden out-of-hospital death included in the Tampere Sudden Death Study. Bacterial DNA was amplified using nested 16S rRNA PCR and sequenced. Amplicon sequence variants (ASVs) were inferred using the DADA2 approach. Taxonomy assignment of the sequences was accomplished using the Silva database (version 138).The presence of bacteria in coronary artery atherosclerotic plaques was confirmed by immunohistochemistry. Results: ASVs from 230 bacteria at the genus level occurring in humans were detected in the coronary plaques. The most common ASVs detected in almost all plaques belonged to the oral biofilm-producing bacteria Veillonella and Streptococcus , which, along with Prevotella, Lactobacillus, Clostridium , and Fusobacterium were the genera with the most ASV reads following nested PCR. The immunopositivity of oral streptococci and Veillonella occurred mainly as biofilm-like structures around calcific plaque areas and associated with the severity of atherosclerosis. Conclusion: Coronary plaques harbor DNA sequences from dozens of mainly oral bacteria. However, only a few bacteria are so common that they likely have a role in the buildup of the microbiome inside a coronary artery atheroma.

Aim: The modest added predictive value of the existing genetic risk scores (GRSs) for coronary artery disease (CAD) could be partly due to missing genetic components, hidden in the genetic architecture of intermediate phenotypes such as coronary artery calcification (CAC). In this study, we investigated the predictive ability of CAC GRS for CAD. Materials and methods: We investigated the association of CAC GRSs with CAD and coronary calcification among the participants in the Ludwigshafen Risk and Cardiovascular Health study (LURIC) (n=2742), the Tampere Vascular Study (TVS) (n=133), and the Tampere Sudden Death Study (TSDS) (n=660) using summary data from the largest multi-ancestry GWAS meta-analysis of CAC to date. Added predictive value of the CAC GRS over the traditional CVD risk factors was tested with standard train–test machine learning approach using the LURIC data, which had the largest sample size. Results: CAC GRS was significantly associated with CAD in LURIC (OR=1.41, 95% CI [1.28–1.55]), TVS (OR=1.79, 95% CI [1.05–3.21]) as well as in TSDS (OR=4.20, 95% CI [1.74–10.52]). CAC GRS showed strong association with calcification areas in left (OR=1.78, 95% CI [1.16-2.74]) and right (OR=1.71, 95% CI [1.98-2.67]) coronary arteries. In addition, there was statistically significant added predictive value of the CAC GRS for CAD over the used traditional CVD risk factors (AUC 0.734 vs 0.717, p-value = 0.02). Conclusions: This study showed that CAC GRS is a new risk marker for CAD in three European cohorts, with added predictive value over the traditional CVD risk factors.

Background: Alcohol use disorder (AUD) is associated with increased mortality and morbidity risk. A reason for this could be accelerated biological aging, which is strongly influenced by disease processes such as inflammation. As recent studies of AUD show changes in DNA methylation and gene expression in neuroinflammation‐related pathways in the brain, biological aging represents a potentially important construct for understanding the adverse effects of substance use disorders. Epigenetic clocks have shown accelerated aging in blood samples from individuals with AUD. However, no systematic evaluation of biological age measures in AUD across different tissues and brain regions has been undertaken.Methods: As markers of biological aging (BioAge markers), we assessed Levine's and Horvath's epigenetic clocks, DNA methylation telomere length (DNAmTL), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in postmortem brain samples from Brodmann Area 9 (BA9), caudate nucleus, and ventral striatum (N = 63–94), and in whole blood samples (N = 179) of individuals with and without AUD. To evaluate the association between AUD status and BioAge markers, we performed linear regression analyses while adjusting for covariates.Results: The majority of BioAge markers were significantly associated with chronological age in all samples. Levine's epigenetic clock and DNAmTL were indicative of accelerated biological aging in AUD in BA9 and whole blood samples, while Horvath's showed the opposite effect in BA9. No significant association of AUD with TL and mtDNAcn was detected. Measured TL and DNAmTL showed only small correlations in blood and none in brain.Conclusions: The present study is the first to simultaneously investigate epigenetic clocks, telomere length, and mtDNAcn in postmortem brain and whole blood samples in individuals with AUD. We found evidence for accelerated biological aging in AUD in blood and brain, as measured by Levine's epigenetic clock, and DNAmTL. Additional studies of different tissues from the same individuals are needed to draw valid conclusions about the congruence of biological aging in blood and brain.

Background: Acute ischemic stroke may be due to embolism from ruptured atherosclerotic carotid arteries. DNA of oral bacteria, mainly the viridans streptococci group, has been detected in thrombus aspirates of patients with ischemic stroke as well as in carotid endarterectomy samples. Because viridans streptococci are known to possess thrombogenic properties, we studied whether their presence in thrombus aspirates and in carotid artery specimens can be confirmed using bacterial immunohistochemistry. Methods and Results: Thrombus aspirates from 61 patients with ischemic stroke (70.5% men; mean age, 66.8 years) treated with mechanical thrombectomy, as well as carotid endarterectomy samples from 20 symptomatic patients (65.0% men; mean age, 66.2 years) and 48 carotid artery samples from nonstroke autopsy cases (62.5% men; mean age, 66.4 years), were immunostained with an antibody cocktail against 3 species ( Streptococcus sanguinis , Streptococcus mitis, and Streptococcus gordonii ) of viridans streptococci. Of the thrombus aspirates, 84.8% were immunopositive for viridans streptococci group bacteria, as were 80.0% of the carotid endarterectomy samples, whereas immunopositivity was observed in 31.3% of the carotid artery samples from nonstroke autopsies. Most streptococci were detected inside neutrophil granulocytes, but there were also remnants of bacterial biofilm as well as free bacterial infiltrates in some samples. Conclusions: Oral streptococci were found in aspirated thrombi of patients with acute ischemic stroke as well as in carotid artery samples. Our results suggest that viridans streptococci group bacteria may play a role in the pathophysiology of ischemic stroke.

Haptoglobin (Hp) is a plasma protein that binds free hemoglobin and protects tissues from oxidative damage. An Hp2 allele has been associated with an increased risk of cardiovascular complications. On the other hand, recent studies have suggested that Hp1 allele increases risk to develop severe cerebral small vessel disease. We aimed to replicate this finding in a first-ever stroke patient cohort. Hp was genotyped by PCR and gel electrophoresis in the Helsinki Stroke Aging Memory Study in patients with DNA and magnetic resonance imaging (MRI) available (SAM; n = 316). Lacunar infarcts and white matter lesions (WML) classified by Fazekas grading from brain MRI were associated with Hp genotypes. As population controls, we used participants of Cardiovascular diseases—a sub study of Health 2000 Survey (n = 1417). In the SAM cohort, 63.0% of Hp1-1 carriers (n = 46), 52.5% of Hp1-2 carriers (n = 141) and 51.2% of Hp2-2 carriers (n = 129) had severe WML (p = 0.372). There was no difference in severe WMLs between Hp1-1 vs. Hp1-2 and Hp2-2 carriers (p = 0.201). In addition, 68.9% of Hp1-1 carriers (n = 45), 58.5% of Hp1-2 carriers (n = 135), and 61.8% of Hp2-2 carriers (n = 126) had one or more lacunar lesions (p = 0.472). There was no difference in the number of patients with at least one lacunar infarct between Hp1-1 vs. Hp1-2 and Hp2-2 groups (p = 0.322). Neither was there any difference when diabetic patients (type I and II) were examined separately. Hp1 allele is not associated with an increased risk for cerebral small vessel disease in a well-characterized Finnish stroke patient cohort.