Exploring lipoprotein characteristics in human cardiometabolic disease

Background: Atherosclerosis underlies most cases of cardiovascular disease (CVD) which remains the principal cause of global mortality. CVD is significantly linked to cardiometabolic diseases such as diabetes and obesity. These disorders precipitate a cascade of metabolic dysregulations, including insulin resistance and altered lipid metabolism, culminating in modified lipoprotein profiles. Aim: Dissecting the characteristics of lipoproteins in the context of cardiometabolic diseases and their contribution to atherogenesis. Methods: The thesis is based upon five papers. I. Assessment of the impact of a novel potential therapeutic agent on hepatic lipid metabolism using modified mRNA molecules in human hepatoma cell lines. II. Characterization of HDL in systemic arterial and coronary blood from patients with Non-ST-Elevation Myocardial Infarction (NSTEMI). III. Evaluation of the influence of clustered cardiometabolic risk factors on the functional properties of HDL. IV. Investigation into mechanisms of lipoprotein retention in type 2 diabetes through analyses of lipoproteins in serum and interstitial fluid. V. Characterization of postprandial lipoproteins in serum and interstitial fluid among healthy controls and individuals with type 2 diabetes. Results: Key findings from the thesis papers were I. The overexpression of TGIF1 protein in hepatoma cells induced a favorable lipid profile alteration. II. Notable differences in HDL properties were found between coronary and systemic circulation in the immediate aftermath of NSTEMI, unrelated to HDL levels. III. Cardiometabolic risk factors exert a progressive negative impact on HDL function, with a major contribution of abdominal obesity to alterations in reverse cholesterol transport. IV. An apparent increase in vascular retention of atherogenic lipoproteins was seen in type 2 diabetes, manifested by reduced ApoB-containing lipoproteins in interstitial fluid concomittant with increased LDL affinity for arterial proteoglycans. Similar changes were observed with increasing age in controls. V. Distinct postprandial alterations in serum and interstitial fluid revealed atherogenic postprandial lipoprotein modifications in Type 2 diabetes. Conclusion: This thesis accentuates the relationship between lipoprotein properties and atherosclerosis in the context of cardiometabolic disorders. The insights highlight the importance of qualitative lipoprotein evaluation beyond conventional quantitative lipoprotein analysis. Such analyses should improve preventive strategies, including cardiovascular risk assessment and new therapeutic interventions

Modifiable risk factors, blood proteins, and venous thromboembolism

Venous thromboembolism (VTE) refers to blood clots in the veins, which is an underappreciated vascular disease that can cause disability and mortality. Although some triggers for VTE (e.g., surgery, fracture, infection, hospitalization, and cancer) have been established, the associations of modifiable risk factors and blood proteins with the risk of VTE remain uncertain. This PhD project aimed to 1) investigate the associations of obesity and lifestyle factors with VTE risk; 2) explore the associations of blood proteins with VTE risk; and 3) establish protein pathways linking modifiable risk factors to VTE development. In Paper I, we explored the associations of overall and central obesity with the risk of VTE using both cohort and Mendelian randomization analyses. We found a potentially causal association between obesity and VTE risk. Waist circumference might be a preferable indicator linking obesity to VTE. Around 12.4% and 23.7% of VTE cases could be prevented if the population maintained a healthy body mass index and waist circumference, respectively. In Paper II, using the prospective cohort design, we investigated the associations of cigarette smoking, alcohol and coffee intake, physical activity, and diet with the risk of incident VTE. We found that high levels of physical activity and a healthy diet were associated with lower VTE risk in women and men. Cigarette smoking showed a positive association with VTE only in women. Alcohol and coffee intake was not associated with VTE. In Paper III, we explored the association between ultra-processed food intake and the risk of VTE using the prospective cohort design. A higher ultra-processed food intake was associated with a moderately increased risk of VTE. This association was not modified by age, sex, or body mass index. In Paper IV, we conducted a prospective cohort and Mendelian randomization study to estimate the associations of 257 blood proteins with VTE risk. The cohort analysis identified 21 blood proteins associated with incident VTE. Machine-learning analysis found that body mass index and von Willebrand factor shared an identical highest ranking concerning the contribution to the prediction model. Mendelian randomization analysis confirmed 7 protein-VTE associations. In Paper V, we performed a two-stage network Mendelian randomization analysis to decipher proteomic pathways underlying the associations of 15 modifiable risk factors with VTE. We found that several proteins, in particular annexin II and coagulation factor XI, mediated the associations of obesity, smoking, and insomnia with VTE. Proteome-wide Mendelian randomization analysis identified many VTE-associated proteins with druggable potentials. In summary, the above five studies identified modifiable risk factors and blood proteins for VTE development and further revealed protein pathways underlying the associations between modifiable risk factors and VTE. These findings may deepen understanding of VTE pathogenesis and facilitate precision prevention and drug development for VTE

Exploring signaling pathways in endothelial mechanotransduction

Mechanotransduction, the process by which cells sense and convert mechanical forces from their surroundings, plays a pivotal role in cellular function. For vascular endothelial cells, coping with the continuous and significant shear stress caused by blood flow is essential for the stability of the circulatory system. Dysfunction in this mechanism can lead to various vascular pathologies, including atherosclerosis and aneurysms. Therefore, understanding how endothelial cells form mechanosensory complexes and thereby respond to external forces is crucial. This thesis aims to study the involvement of the Angiomotin protein family in endothelial mechanotransduction. The Amot protein family, including Angiomotin (Amot), Angiomotin-Like 1 (AmotL1), and Angiomotin-like 2 (AmotL2), shares common structures as well as protein interaction motifs. However, they exhibit significantly different roles in vascular functions. In Paper I, we elucidate how Amot binds to Talin within the integrin adhesome and regulates force transmission between fibronectin and the cytoskeleton in migrating endothelial cells. Additionally, we demonstrate that deletion of Amot impairs both physiological and pathological angiogenesis. In Paper II, AmotL2 isshown to bind VE cadherin through p120 catenin and connect to the nuclear membrane via actin filaments in aortic endothelial cells, thereby transmitting junctional mechanical signals. Depletion of AmotL2 resulted in a pro-inflammatory response and, in severe cases, leads to the spontaneous formation abdominal aortic aneurysms (AAAs) in male adult mice. In Paper III, it is demonstrated that AmotL1 not only binds to N-cadherin but is also associated with focal adhesion proteins. This suggests that AmotL1 functions may extend beyond endothelial cell junctions to include interactions with the extracellular matrix. Additionally, we provide a comprehensive summary of the protein binding profiles of all Amot proteins, as obtained from BioID-MS analysis, thus offering a global perspective on this protein family. In conclusion, Amot family proteins, despite their involvement in separate cellular processes, share common connections with a set of junction-related proteins. Furthermore, they exhibit unique, specific binding partners, offering mechanistic insights into the distinct activities of individual Amot proteins

Skeletal muscle responses to physical activity in health and metabolic disease

Sedentary lifestyles, characterised by a lack of physical activity and prolonged periods of sitting, have been linked to reductions in whole-body metabolic flexibility and the increased risk of metabolic diseases, including type 2 diabetes. This can be attributed, at least partially, to the direct negative effects of physical inactivity on skeletal muscle insulin sensitivity, oxidative capacity, and overall metabolic health. In addition, sedentary behaviour can lead to anabolic resistance, resulting in losses of skeletal muscle mass and strength, which can further contribute to conditions like sarcopenic obesity, impairing physical performance and overall quality of life. Conversely, physical activity plays a crucial role in maintaining and improving skeletal muscle health. Exercise is associated with various adaptations in skeletal muscle that enhance tissue oxidative capacity, substrate handling, insulin sensitivity, as well as skeletal muscle mass and strength. These positive changes in skeletal muscle contribute to improvements in systemic metabolic wellbeing. The molecular mechanisms underlying skeletal muscle adaptation to the perturbations caused by physical activity are complex and involve intrinsic processes within the muscle fibre itself, as well as communication between different cell populations in composite skeletal muscle tissue. However, our understanding of the intricate details of these mechanisms remains incomplete. Gaining deeper insights into the regulation of skeletal muscle adaptation could not only facilitate personalised exercise recommendations but also uncover novel opportunities for drug discovery, ultimately leading to improvements in human health. Despite the well-known benefits of exercise, physical activity guidelines are often not met by the general population. Therefore, there is a pressing need for low-level entry paradigms that can promote physical activity and reduce sedentary behaviour for the betterment of individual and public health. One such approach is the incorporation of frequent activity breaks or ‘exercise snacks’ into daily routines, which involves short-duration physical activity breaks throughout the day to disrupt prolonged periods of sitting. These interventions have demonstrated efficacy for cardiometabolic health in controlled settings, such as laboratory-based clinical trials. However, it is essential to evaluate the benefits of breaking sedentary time using strategies that better mimic real-world scenarios to inform practical public health guidelines. In this thesis, the following objectives were pursued: (1) To assess the translational efficacy of interrupting sedentary time in improving cardiometabolic health. (2) To investigate the skeletal muscle transcriptome following exercise or physical inactivity in the context of health and metabolic diseases. (3) To determine the metabolic effects of physical activity- responsive transcription factors and signalling molecules in skeletal muscle. Study I revealed that even a minor addition of ≈750 steps dispersed throughout the day, equivalent to ≈10 minutes of extra walking time, improved dynamic glucose control in individuals with obesity and insulin resistance. Notably, those who engaged in higher levels of physical activity while interrupting sedentary time experienced greater benefits, indicating that more breaks from sedentary behaviour lead to better metabolic health outcomes. Nevertheless, adherence to the intervention, which involved 3-min activity bouts every 30 min between 08:00-18:00, was lower than anticipated. This raises questions about the long- term feasibility of such approaches when considered in isolation from other modifiable lifestyle factors, including changes in dietary habits or structured exercise routines. Study II employed a comprehensive meta-analytical approach to compare the skeletal muscle transcriptomic response to acute aerobic or resistance exercise, exercise training, and physical inactivity. This analysis revealed distinct gene signatures in skeletal muscle after a single bout of exercise in the naïve state, which differed from those observed after training of the same exercise modality. Interestingly, there was greater overlap in the skeletal muscle transcriptome between acute aerobic and resistance exercise than there was between acute exercise and exercise training. These findings highlight the refinement of the adaptive response in skeletal muscle over time through dedicated training to a specific exercise modality. Study II identified the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3) as a gene that is upregulated in skeletal muscle after exercise but downregulated in response to physical inactivity. Study III delved deeper into the role of NR4A3 in the context of physical inactivity and revealed its regulatory role in translation within skeletal muscle. Depletion of NR4A3 resulted in skeletal muscle atrophy and compromised glucose oxidation, instead favouring increased lactate production. Therefore, decreased levels of NR4A3 during physical inactivity may directly contribute to muscle disuse atrophy and impaired skeletal muscle metabolism. Furthermore, study II identified that individuals with obesity and/or type 2 diabetes exhibit an altered skeletal muscle transcriptional response to exercise training compared to healthy individuals. Study IV uncovered a heightened inflammatory response during the recovery period after exercise in individuals with type 2 diabetes. This response was attributed to an increased influx of immune cells into skeletal muscle tissue, potentially facilitating crosstalk between different cell types within the skeletal muscle interstitial space. Notably, the cytokine stromal cell-derived factor 1 (CXCL12/SDF-1) was found to be expressed by macrophages or endothelial cells in response to factors released by skeletal muscle fibres or hypoxia, respectively. CXCL12 activation, in turn, promoted the proliferation of skeletal muscle satellite cells, which could be integral for adaptive remodelling following exercise. In conclusion, the research presented in this thesis emphasises the central role of physical activity in improving human health, with a specific focus on the ability of exercise to induce adaptations in skeletal muscle. The findings herein shed light on the intricate molecular mechanisms underlying skeletal muscle responses to physical activity, which contribute to the metabolic fitness of this tissue and of the human body as a whole

Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.Knut and Alice Wallenberg Foundation (2019-0059)Swedish Research Council (2019-1209, 2017-06014)Swedish Association against RheumatismErling Persson Foundation (2017-10-09)German Federal Ministry of Education and Research (GO-Bio-project aidCURE; 031A385)Federal State of Hesse (LOEWE-project 13, IME Fraunhofer Project Group TMP at Goethe University)Fraunhofer Cluster of Excellence for Immune-Mediated Diseases CIMDSpanish Ministry of Universities through the European Union (NextGeneration EU)Publishe

Advancements in detection of performance enhancing drugs in dried blood spots : focusing on erythropoietin

The use of erythropoietin (EPO) for performance enhancing purposes is detected mainly in urine and serum samples in anti-doping laboratories. However, dried blood spots (DBS) have emerged as a potential additional sample for EPO and other various doping substances. The aims of these studies were to develop a method for EPO analysis from DBS (study I) and use this method to sensitively detect endogenous EPO, micro-doses of exogenous EPO, and the EPO c.577del protein VAR-EPO (studies I-III). I also observed the stability of EPO and insulin-like growth factor 1 (IGF-I) in DBS (study I and V), along with investigations into the relationship between EPO and testosterone (study IV). Sample collection: Capillary blood (for DBS), venous blood (for DBS and serum) and urine were collected from healthy volunteers. The DBS devices used were Capitainer®B 50, Mitra® VAMS, Tasso-M20, and Whatman 903 filter paper cards for EPO and IGF-I detection. Urine and serum were also collected from 30 self-reported anabolic androgenic steroid (AAS) users to examine the presence of EPO in such samples. Methods: The primary method I used for detecting EPO and VAR-EPO in the collected samples was immunopurification with the EPO Purification Gel Kit, SAR- PAGE, and Western blot. EPO concentrations were measured in serum using a commercial immunoassay kit. IGF-I was analyzed by LC-MS/MS and with the automated immunoassay system IDS-iSYS. Serum AAS was quantified using an immunoassay. Results: Endogenous EPO was sensitively detected on both polymer and paper blood supports (studies I, II, III), providing stable results (study I), while urinary EPO fluctuated more (study I) and occasionally showed degradation or undetectable bands (studies I and III). The instability of IGF-I in DBS at room temperature (study V) and the slight variations of EPO detection indicate that currently DBS is not suited for longitudinally monitoring these markers. In addition, micro-doses of various recombinant EPOs (rEPO) and VAR-EPO were well-detected in 4 DBS devices from healthy volunteers (studies II, III). When rEPO micro-doses were administered with testosterone to healthy volunteers, as expected, there was no increase in EPO signal intensity in DBS, but in AAS users who were positive for testosterone, serum EPO concentrations were slightly elevated, along with some hematological parameters (study IV). Conclusion: The detection and knowledge of EPO in DBS has made progress, but DBS detection methods, sample collection, and storage strategies still require further discussion and investigation

Network and behavioral correlates of prefrontal neurons

The prefrontal cortex is located in the front of the brain. This region is considered crucial for cognitive processes, such as decision-making, attention, and working memory. Neuronal activities in this region are found to correlate with cognitive features, as well as behavioral variables such as location and movement. Within the rodent brain, there are distinct neuron types. Research indicates that the function of neurons, whether in relation to local networks or to behavior, is to some extent cell-type-specific. In this thesis I explore methods to study neuronal cell-types in the rodent brain. Further, I use these methods to investigate the role of parvalbumin (PV) expressing interneurons in rats, in relation to network dynamics and behavioral features. This thesis contains 2 papers. Paper I: We developed and described a method for concurrent electrophysiological recordings and optogenetic manipulation in freely moving rodents. Specifically, we designed a low-cost microdrive system and demonstrated its utility in freely moving rats and mice. Paper II: We presented and characterized a novel PV-Cre rat line. We used this rat line to study the activity of prefrontal neuronal subpopulations in rats performing a goal-directed reward-seeking task. Consistent with previous findings, our data reveal neuronal tuning to both spatial and movement variables, with the strongest tuning observed for linear position. Additionally, in a subset of neurons, we observe activities that correlated with the conjunction of location and movement direction, referred to as the spatial context. While the activity of single neurons of all types were correlated with the spatial context, it was most prominently observed in the PV interneuron population

Child health in the Democratic Republic of the Congo : exploring subnational disparities and inequities in illnesses and mortality

Background: Today, sub-Saharan Africa accounts for a disproportionally high burden of deaths in children, with the Democratic Republic of the Congo (DRC) being one of the countries with the highest mortality burden. Ample evidence exists on what protective, preventive, and curative services need to be in place to end preventable deaths in children. However, context-specific sub-national data that can effectively guide public health interventions is scarce for the DRC. This thesis first explored causes of death and coverage of preventive interventions to give a broad understanding of inequities around child mortality. Based on these findings, a case study approach was taken to look in depth at system factors that contribute to diarrhoeal deaths. Methods: In Paper I, household survey and health facility data were used to compare child mortality rates and coverage of key indicators known to lower child mortality at the provincial level. Conflict event data was further used to classify provinces according to conflict intensity to explore conflict exposure as a predictor for child health. Paper II used demographic and verbal autopsy data to empirically estimate the causes of and circumstances around deaths. Papers III and IV used focus group discussions, health facility audits, and knowledge questionnaires to understand the health system’s capacity for cholera surveillance and diarrhoeal disease case management in cholera hotspots. Results: Paper I identified large disparities in child mortality and indicator coverages. Conflict was found to be a poor predictor of child health. Provinces classified as conflict-affected had both the highest and lowest under-five mortality rates and indicator coverages. Paper II (manuscript) identified infectious diseases as the main cause of death in children and adolescents 1month to 19 years of age. Most deaths occurred at home; however, the majority sought care in the days preceding death. Papers III and IV found low capacity for diarrhoea surveillance and case management, and that health facility providers, drug shop vendors, and traditional health practitioners are perceived as important providers of care and should be considered in health policy plans. Conclusions: Sustained, and more equitable, health system strengthening efforts are needed to decrease sub-national disparities and end preventable deaths in children in the DRC

Developing an embryonic stem cell-based cell product for age-related macular degeneration

Age-related macular degeneration (AMD) remains a major cause of blindness with no current cure. With the increasing ageing population in the developed world, a high number of AMD patients is expected. AMD can be distinguished between dry or wet AMD. 10-15% of patients present wet AMD which can be treated with anti-VEGF injections. In contrast, dry AMD management has just recently advanced towards a new drug to treat and to halt the progression of the disease by targeting the complement system. However, we are still far from cure of AMD and more efforts are needed towards development of stable and long-term treatment. With the lack of conventional drug options, the field has moved into the investigation of stem cell-derived therapies. In the case of AMD, the cells that degenerate are retinal pigment epithelium (RPE) cells. For a decade, the lab has worked towards the development of a xeno-free, chemically defined, and scalable protocol to generate stem cell-derived RPE. This is achieved whereas translation into the clinic remains. Within my PhD studies, I have performed the following studies: Firstly, by using single-cell RNA sequencing (scRNAseq) we explored the different cell types that emerged during in vitro differentiation towards hESC-RPE at various time points. The transcriptomic profiling showed a diversity of cells recapitulating the early embryonic development in the first weeks of gestation, showing genetic expression of neural crest, placodal and mesenchyme. The differentiation protocol has a replating step in the middle of the protocol, that induced a rapid move of the cell’s profile towards a pure RPE population which by the end of the protocol matured further. At the replating step, cells could be driven to other lineages with the selection of NCAM1 positive population showing the potential of this retinal progenitor’s marker. Thus generated mature, pure, and functional hPSC-RPE were transplanted into rabbit’s eyes, a largeeyed animal model. After 28 days, transplanted cells were retrieved and scRNAseq was performed revealing that those cells were able to mature even further in vivo. Secondly, cryopreservation offers a highly important step in the manufacturing process of a cell product. Cryostorage offers flexibility between the manufacturing location, the testing of the product, and the patient availability. It also confers the ability to produce larger batches, reducing the costs of manufacturing. After the discovery of the incapability of our hPSC-RPE to cryopreserve, we introduced and optimized an extra replating step, regarding time and densities, to be able to successfully cryopreserve our drug product. This replating step didn’t impact the identity of the hPSC-RPE, since after preservation it had characteristic RPE traits and preserved functionality. Transcriptionally, the replated hPSC-RPE presented a cycling profile and a shrinker maturation profile compared to the terminally mature hPSC-RPE obtained in the original protocol. A noninvasive tool is presented to track the optimal replating time window before cryopreservation based on brightfield images based on the cobblestone morphology of the cells. Lastly, we addressed translation towards the clinic. We proceeded to good manufacturing practice (GMP) adaptation of our protocol that introduced slight differences to improve cost-effectiveness, scale up the manufacture, and introduce large-batch instruments into our process. We successfully completed one engineering and one GMP batch in the Cell Therapy Center of Karolinska University Hospital and cryopreserved them. Most have been used in the extensive release testing, stability program, and preclinical testing presented here. Conventional release testing was complemented with newly developed and validated assays including a more sensitive in vitro assay for lingering pluripotent stem cells or transformed cells. Dose precision and in-use stability studies were performed to mimic the surgical setting for suspension injections. Route of administration feasibility was tested in nude rats, evaluating subretinal injection through transscleral or transvitreal route, revealing that the latter minimized the risk of procedure-related complications. The 28-days toxicology studies in nude rats showed no human cells outside the eyes. No unexpected pathological outgrowth or clinical symptoms were observed in the nude rats dosed with the drug product. Efficacy studies in Royal College of Surgeons (RCS) rats for 90 days after injections show reduced degeneration of the neuroretina of the rats. Optokinetic response and electroretinography showed higher light sensitivity in dosed animals than in vehicles, showing functionality of the implant. Further application to the Swedish Medical Agency is planned for late this year. Taken together, the studies presented in this thesis represent a step forward toward the first stem cell-based therapy to treat AMD in Sweden. I) An unbiased characterization of our hPSC-RPE differentiation protocol is presented showing all cellular composition through the different stages by scRNAseq. II) An optimization step to produce large batches and cryostore the hPSC-RPE cells for flexible manufacturing and delivery of cells. III) Our drug product, CellThRPE1, has been extensively tested and is ready for regulatory review prior to entering the clinical phase

Testosterone is performance enhancing in women but challenging to detect in doping analyses

BACKGROUND: There is an ongoing debate whether women with testosterone (T) within the male range shall be allowed to compete in the female class in sports. In men, T has an ergogenic effect, but the effect of T in young women has not been investigated previously. We hypothesised that increased levels of T would enhance physical performance in exercising women. T is classified as a doping agent by the World Anti-Doping agency (WADA). However, the existing doping analysing methods seems to be insufficient to detect exogenous use of T in women. In addition, the menstrual cycle and the use of hormonal contraception might aggravate the interpretation of the doping test results. Accordingly, the percentage of positive doping tests are higher in men than in women. The aim was to study the effect of T in female athletic performance, as well as endocrine influence on the steroid profile of relevance for anti-doping testing in women. METHODS: The studies in this thesis are based on two double-blind randomised, placebo-controlled trials. Forty-eight young healthy women were randomised to 10 weeks of 10mg daily T-cream or placebo to study the effects of moderately increased T concentration on physical performance, body composition, psychological well-being and self-confidence, and the steroid profile in urine and blood. Physical performance was measured by performance tests at the Swedish School of Sports and Health Sciences. Body composition was measured with DEXA. Well-being and self-confidence were measured by questionnaires. We also studied the effect of combined oral contraceptives (COC) and the menstrual cycle phases and genetic factors on the steroid profile using serum and urine samples from a trial where 348 women were randomised to three months of COC or placebo. Genotyping, immunoassays, LC-MS/MS and GC-MS/MS were performed. MAIN RESULTS AND CONCLUSIONS: Our findings support a causal effect of T on physical performance as measured by an increase in running time to exhaustion of 8.5% in young healthy women. T also promotes an increase in lean mass and might influence phycological well-being and confidence. The results are of importance for the understanding of the sex difference in athletic performance, as well as for regulations of hyperandrogenism in women’s sports. Standard anti-doping urine analyses detected T-administration in only two out of 24 participants. Individual thresholds increased the detection rate to 40%. Furthermore, it was shown that COC have great impact on the urinary steroid biomarkers included in anti-doping analyses. COC suppressed the serum steroids and phase II metabolites while the ratio of T and androstenedione (A4) remained stable. Moreover, the results confirm that T/A4 remained stable throughout the menstrual cycle. T-administration increased the T/A4-ratio in serum 3.5 times compared to placebo. We can conclude that the urine analyses in use today is insufficient to detect T-doping in women. Instead, we suggest serum testing, including the ratio of T/A4