WO2020165388A1

WO2020165388A1 - Method for monitoring, evaluating or improving the fitness by analysis of mirna markers

Info

Publication number: WO2020165388A1
Application number: PCT/EP2020/053840
Authority: WO
Inventors: Alexander Haslberger; Juerg Daniel Schmid; Ulrike KRAMMER
Original assignee: System Biologie Ag
Priority date: 2019-02-15
Filing date: 2020-02-14
Publication date: 2020-08-20

Abstract

Subject of the invention is a method for monitoring, evaluating and/or improving the fitness of an individual, comprising the steps of (a) providing a first sample, which is a body fluid or fraction thereof obtained from the individual at a first point in time, (b) providing a second sample, which is the same body fluid or fraction thereof obtained from the individual at a second point in time, (c) determining and comparing with each other the levels of at least one microRNA marker in the first and second sample, and (d) monitoring, evaluating, and/or improving the fitness of the individual based on the result of the comparison made in step (c). The invention also relates to a system for using the method and uses of microRNA markers.

Description

Method for monitoring, evaluating or improving the fitness by analysis

of miRNA markers

The invention relates to a method for monitoring, evaluating and/or improving the fitness of an individual by comparative analysis of the level of at least one miRNA marker, and to a system for carrying out the method.

State of the art

There is a growing interest in reliable and objective information about the fitness of individuals. The fitness of an individual includes long-term mental and physical fitness, and thus in fact general well-being and health. Information about the fitness is frequently obtained by non-invasive methods, such as determination of pulse, blood pressure, motor capability and body-mass index, by electrocardiography and the like. However, such non- invasive methods are relatively superficial, depend from external factors, and are thus not always reliable. Non-invasive parameters often tend to describe symptoms rather than physiological fundamentals. Therefore, the focus of attention is shifting towards methods in which specific biomarkers of the individual are analyzed in body fluids. Levels and changes of biomarkers in blood, sputum or urine, may relate to general health and fitness of individuals. For example, such biomarkers could be proteins, metabolites, electrolytes or other small molecules in body liquids.

US 8,762,167 B2 discloses methods for establishing personal health plans based on biomarkers. A vast number of potential biomarkers is suggested which could be analyzed. However, the method is unspecific and of limited value for recommendations for an individual. The document rather shows that there is a need for new, specific methods and tools for such purposes.

Lee et al. , 2017, is a review article about biomarkers related to sports and exercise. However, as noted in this document, the physiological interactions which affect levels of such biomarkers are relatively complicated. Therefore, there is an ongoing need for new precise and reliable biomarkers for such purposes.

In the last years, there has been growing attention on microRNAs as potential biomarkers. miRNAs (microRNA, micro-ribonucleic acid) are small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses that function in RNA silencing and post-transcriptional regulation of gene expression. They are mediators of various biological processes and can be detected in body liquids of humans. However, the specific functions of most miRNAs are still poorly understood. Polakovicova et al. , 2016, provides an overview on publications regarding miRNAs as potential biomarkers of exercise response. Respective scientific publications are summarized in table 1 thereof, whereas miRNAs for which effects have been described are summarized in table 2. Most scientific studies relate to short-term effects. For example, samples were taken during or after strenuous exercises. However, the findings are diverse and seem to depend from the specific conditions of the exercise. Therefore, it would be relatively difficult to provide specific recommendations for individuals based on the data included.

Fernandez-Sanjurjo et al., 2016, provides a review on publications on miRNA as regulators in the molecular response in exercise in healthy people. Initially, it is noted that various papers have published inconsistent results. The tables in the document show that almost all publications in this field relate to changes of miRNA levels after acute exercise and immediately after exercise.

Domanska-Senderowska et al., 2017, describes a study on whether the expression of three pre-determined miRNA in blood serum changes during prolonged soccer training. The authors found that the expression of miR-29a is increased over time, whereas the other two miRNA levels are not affected.

Baggish et al., 2011 , describes some miRNAs which were found up- or down-regulated in blood plasma samples from individuals after acute exhaustive exercise and sustained aerobic exercise training. Davidson et al., 2011 , relates to the expression of miRNAs in skeletal muscle. Samples were taken from individuals by needle muscle biopsies after frequent and strenuous exercise. Such documents focus on physiological pathways which affect miRNAs after acute exercise. There is an ongoing need in the prior art to provide new methods for monitoring, evaluating or improving the fitness of an individual, which are simple, reliable and precise.

Problem underlying the invention

The problem underlying the present invention is to provide methods for monitoring, evaluating and improving the fitness of an individual, which overcome the above mentioned problems. It would be desirable to provide new methods which are reliable, precise and relatively easy and convenient to perform. It is a specific problem of the invention to provide such improved methods based on the analysis of biomarkers, such as miRNAs. Disclosure of the invention

Surprisingly, it was found that the problem underlying the invention is overcome by methods, systems and uses according to the claims. Further embodiments of the invention are outlined throughout the description.

Subject of the invention is a method for monitoring, evaluating and/or improving the fitness of an individual, comprising the steps of

(a) providing a first sample, which is a body fluid or fraction thereof obtained from the individual at a first point in time,

(b) providing a second sample, which is the same body fluid or fraction thereof obtained from the individual at a second point in time, (c) determining and comparing with each other the levels of at least one miRNA marker in the first and second sample, and

(d) monitoring, evaluating, and/or improving the fitness of the individual based on the result of the comparison made in step (c), wherein the at least one miRNA marker is selected from the group of miR-505, miR-22, miR-30, miR-19, miR-101 , miR-378, miR-494, miR-369, miR-122 and miR-135. The individual is a human individual. In one aspect, the method is for monitoring the fitness. In this embodiment, data is gathered in step (c) which can be used for evaluating and/or improving the fitness. In another aspect, the method is for evaluating the fitness. This means that a qualitative or quantitative conclusion regarding the fitness of the individual is made based on data gathered in step (c). In another aspect, the method is for improving the fitness. In this embodiment, the method comprises additional measures for improving the fitness of the individual, such as providing recommendations for improving the fitness to the individual, for example by an exercise plan and/or a diet.

As used herein, the term "fitness" comprises the physical and mental fitness of the individual. Generally,“fitness” relates to a general state of health and well-being. This includes the ability to perform sports, occupations and daily activities. Fitness is generally achieved through proper nutrition, moderate physical exercise and sufficient rest. Fitness is a basic long-term state of the individual. Changes of fitness usually require extended time periods, for example in the range of weeks or months. Thus, fitness is different from a temporary condition, for example on which changes within hours or a few days, such as weariness after exercise.

In one embodiment, the fitness is the physical fitness. Physical fitness is especially associated with the capability to carry out physical exercise and/or sport.

Fitness also relates to a state of well-being. As used herein, the term "well-being" refers to the general condition of the individual. Specifically, a high level of well-being means that the individual's basic condition is positive.

Fitness also relates to the general health of the individual. As used herein, the term "general health" relates to a state of complete physical, mental and social well-being. It does not merely imply that a disease is absent. Therefore, the method is different from other methods, which relate to therapeutic treatment of a specific disease. Preferably, the inventive method does not encompass and/or is not related to diagnosis or treatment of a disease.

In the inventive method, there is no specific order of steps (a) and (b). Step (c) of determining and comparing the levels of markers is carried out after steps (a) and (b) with the first and second sample provided in steps (a) and (b). In step (c), data is obtained from the first and second sample. Subsequently, the data is used for monitoring, evaluating and/or improving the fitness in step (d).

The individual is a human individual. In a preferred embodiment, the individual is a healthy individual and/or does not suffer from a chronic disease. In a preferred embodiment, the individual is a healthy individual. Preferably, the individual does not suffer from a disease, especially a severe disease or chronic disease; or does not suffer from a disease which affects the long-term fitness. In contrast, it is not detrimental to the method if the individual experiences a temporary disorder, such as a cold or influenza, for a limited time span, for example for less than 2 weeks, or less than 1 week. The individual will recover the previous fitness after such a temporary disorder has been overcome. In this case, the method can be interrupted such that no data is gathered during the time span. Alternatively, the method may proceed during the time span, but the disorder is taken into account when the data is evaluated.

In a specific embodiment, the age of the individual can be below 70, below 60 or below 50 years. The individual may be an adult. The age of the individual may be between 18 and 70 or between 18 and 60 years. Specifically, the age could be between 30 and 50 years. The individual could be male or female. In a preferred embodiment, the individual is male. It was found that the changes in the levels or some miRNA markers can be especially significant for male individuals.

In a preferred embodiment, the individual changed his/her lifestyle between the first and second point in time. Preferably, the change of lifestyle is expected to improve the fitness, such as a physical exercise program, a diet, a reduction of drugs and/or regular motion. Preferably, the change of lifestyle is purposeful, for example according to a plan or recommendations provided to the individual. Preferably, the method comprises providing such a plan or recommendations to the individual, for example by a fitness advisor. In these embodiments, it can be determined by the method if the change of lifestyle had an effect on the fitness, and preferably if the change of lifestyle has improved the fitness of the individual.

Herein, the overall, long-term change of lifestyle is also referred to as the“intervention”. For example, the intervention can be a sport program with repeated exercise over several weeks, or a change of diet for several weeks. In this case, the beginning of the intervention is the start of the diet or exercise program, whereas the end of the intervention is the endpoint of the diet or exercise program.

The first and second samples are obtained from the individual at different points of time. By comparison of the results from the first and second sample in step (c), it can then be determined whether the change in lifestyle had an effect on fitness. Preferably, this invention affects the fitness and/or is expected to affect the fitness. For example, the individual has carried out activities, been subjected to a treatment, or changed the nutrition.

In a preferred embodiment, the individual carries out a physical exercise program between the first and second point in time. Preferably, the physical exercise program was moderate and/or a leisure exercise program. For example, a moderate or leisure exercise is one in which the total energy consumption is between 150 kcal and 600 kcal, specifically between 180 kcal and 500 kcal, per day and/or in a single unit. The program usually comprises multiple units at regular time intervals, wherein typically not more than one unit is carried out per day. The unit can be one of standard leisure sport, such as a fitness center workout, jogging, cycling, hiking, swimming, gymnastics and/or ball and team sports. The physical exercise program typically comprises multiple exercise units per week, for example two, three or four times per week. Preferably, the individual is not a professional athlete. Preferably, the individual normally does not carry out physical exercise for more than three or four times per week.

In another preferred embodiment, the individual changed his/her diet between the first and second point in time. In this regard, the term "diet" is not limited to a reduction of the uptake of energy or food, but generally refers to any directed changes of the food uptake.

In another embodiment, the change of lifestyle comprises reduction of drugs, such as consumption of alcohol or tobacco. In another embodiment, the change of lifestyle comprises regular motion, for example walking, hiking, household work or reduction of seated activities.

The length of the time interval between the first and second point in time is not particularly limited. In principle, any time period can be chosen, throughout which a basic change of the fitness of the individual may be achieved. Typically, such a change of fitness will require a reasonable time span at least in the range of weeks. In a preferred embodiment, the time period between the first and second point in time is at least one week, preferably at least two weeks or most preferably at least one month. The time period could be less than 5 years, less than 1 year or less than 6 months. Specifically, the time period could be between 1 week and 5 years, or between 2 weeks and 1 year, preferably between 4 and 60 weeks.

When the time period is relatively long, it is preferred to obtain further samples from the individual in order to monitor not only a starting and end point, but also a progress of fitness during the time period. In a preferred embodiment, the method is carried out also with additional samples, which are obtained from the individual at other time points between the first and second point in time. For example, the method can be carried out with a total of 2, 3, 4, 5, up to 10, or even more samples. In the method, the samples may be obtained weekly, monthly or at other desired time intervals. This is advantageous, because more precise monitoring is possible based on multiple samples.

The method is used especially for monitoring changes of basic, ling-term fitness. Thus, it should be carried out such that changes of the temporary condition of the individual do not distort the result. For example, it is preferred that the samples are not taken from the individual during or shortly after exercise, because then undesired temporary and short term changes of miRNAs may be detected. Thus, it is generally preferred that the samples are taken for a sufficiently long time period after exercise, such as at least 2 hours, preferably at least 12 hours. More preferably, the samples are taken at least 48 hours or at least 72 hours after exercise, especially the final sample (at time point T1) which is taken after the intervention. It is most preferred to take the final sample (T1) at about 72 hours after the final exercise, for example between 3 and 14 days, or between 4 and 10 days. Similarly, other external temporary interventions should be avoided which could affect the result. Thus, it is generally preferred to take the last sample at a time point at which temporary effects of the intervention are negligible. Generally, it would be sufficient to take the last time point at least 2, 3 or 4 days after the end of the intervention. Specifically, it is preferred that all samples are taken under comparable, similar external conditions. Then, that it can be expected that differences of miRNA levels between samples are not caused by external circumstances, which do not relate to fitness. In a preferred embodiment, the body fluid is blood, saliva and/or urine. These body fluids can be obtained easily from the individual. It is most preferred that the body fluid is blood or saliva. In further embodiments, a fraction of a body fluid can be used, such as blood plasma, blood serum or the like.

Preferably, the body fluid was obtained non-invasively or minimally invasively. This is advantageous, because the sample can be obtained in a simple manner by the individual himself, without participation of medical staff. Saliva or urine is obtainable non-invasively. As used herein, a minimally invasive method is not a surgical method. For example, a small drop of blood can be obtained non-invasively, for example from a simple finger prick. In a preferred embodiment, the method comprises a preceding step in which the body fluid is obtained non-invasively or minimally invasively, preferably by the individual.

In a highly preferred embodiment, the samples are dried blood spots (DBS). Dried blood spot testing is a form of biosampling where blood samples are blotted and dried on filter paper. In particular, samples-drops of whole blood can be collected on filter paper from a simple finger prick (capillary blood). The dried samples can easily be shipped to an analytical laboratory and analyzed using various methods such as DNA amplification or HPLC. The use of DBS is advantageous for the present method because dried blood spots are easy to obtain, require only a very low amount of blood and can be transported, stored and processed conveniently. Moreover, it was found that levels of specific miRNAs in dried blood spots provide significant information about the fitness of individuals.

In the inventive method, the levels of at least one miRNA marker in the first and second samples are determined and compared. Preferably, the comparison is a quantitative comparison, in which absolute levels of markers are determined and the difference is calculated. Such an analysis is possible by standard methods. Typically, miRNAs in a sample are amplified by PCR technology. For detection specific miRNA, analytic methods based on two-dimensional arrays are known and established. Typically, miRNAs can be quantified in a two-step polymerized chain reaction process, which combines real-time PCR (RT-PCR) and quantitative PCR. The absolute and relative amounts of miRNAs can be determined. miRNAs can be analyzed by hybridization to microarrays, slides or chips, which comprise probes of hundreds or thousands of targets. For example, tools and services are provided by Qiagen, DE. MicroRNAs (miRNA) are small, non-coding RNA molecules, which typically contain about 22 nucleotides. They are detectable in human body fluids, such as saliva, blood and urine. Herein, miRNAs are named according to the standard nomenclature system. The prefix "miR-" refers to a mature form of a miRNA. The subsequent number identifies the specific miRNA in the order of their discovery. Minor sequence variations of the same miRNA are marked by an additional lower case letter, such as miR-146a. Herein, the absence of such a lower case letter means that all variants of the miR number are comprised. For example, when the present disclosure cites miR-146, this includes miR-146a and miR-146b. The species form which the miRNAs originate can be indicated by another prefix, for example “hsa-“ for Homo sapiens.

At least one miRNA marker, which is analyzed in the method, is selected from the group of miR-505, miR-22, miR-30, miR-19, miR-101 , miR-378, miR-494, miR-369, miR-122 and miR-135. Preferably, at least 2 markers, preferably at least 3 markers, at least 5 markers or at least 8 markers from this group of miRNAs are used in the method. Preferably, between 2 to 10 markers of are used, more preferably between 3 and 10, between 5 and 10 or between 5 and 10 of these markers. In a preferred embodiment, all markers of the group are used.

More preferably, at least one miRNA marker is selected from the group of miR-505, miR-22, miR-30e, miR-19b, miR-101 , miR-378a, miR-494, miR-369, miR-122 and miR-135a. It was found that especially the levels of these variants of the miRNAs were changed. More preferably, at least one miRNA marker is selected from the group of miR-505-3p, miR-22-5p, miR-30e-3p, miR-19b-3p, miR-101-3p, miR-378a-3p, miR-494- 3p, miR-369-5p, miR-122-5p and miR-135a-5p. It was found that the levels of these strands of the miRNAs were changed. Preferably, at least 2 markers, preferably at least 3 markers, at least 5 markers or at least 8 markers from this group of miRNAs are used in the method. Preferably, between 2 to 10 markers of this group are used, more preferably between 3 and 10, between 5 and 10 or between 5 and 10 of these markers. In a preferred embodiment, all markers of the group are used. In a preferred embodiment, at least one miRNA marker is selected from the group of miR-505, miR-22, miR-30, miR-19, miR-101 and miR-378. Preferably, the variants and/or strands are selected as defined above. Preferably, at least 2 markers, at least 3 markers or at least 4 markers from this group of miRNAs are used in the method. In a preferred embodiment, all markers of the group are used. It was found that the levels of these miRNAs changed especially significantly in the inventive method. Surprisingly, it was found that the levels of these specific miRNAs change significantly when individuals carry out physical exercise regularly for a prolonged time. The changes of the marker levels are highly significant or at least significant for all these markers. Therefore, the markers relate to the general fitness of the individuals. These markers have not been identified in the prior art for monitoring, evaluating or improving fitness.

In a preferred embodiment, at least one miRNA marker is selected from the group of miR- 505, miR-22 and miR-30e. In a highly preferred embodiment, the miRNA marker is miR- 505. In another highly preferred embodiment, at least miR-22 is selected. In a highly preferred embodiment, at least miR-505 and miR-22 are selected. Preferably, at least one or two miRNA markers are selected from the group of miR-30 and miR-22. Preferably, the variants and/or strands are selected as defined above. It was found that especially these markers are significant for determining changes in fitness, and that especially these markers are highly significant. These markers can be used exclusively, but it is more preferred to use them in combination with other markers described herein. When multiple markers are used, the result is generally more reliable.

The markers listed above can be used in steps (c) and (d) of the methods. In other words, the levels of these markers are determined and compared in step (c) and the monitoring, evaluating or improving of the fitness in step (d) are based on the comparison. Thus, the method is different from other known methods, in which the levels of a high number of miRNAs in a sample is simply determined, and possibly also compared, but in which the results of the comparison are not used purposefully for monitoring, evaluating, and/or improving the fitness.

In an embodiment of the invention, improving the fitness correlates with a higher level of at least one of the miRNAs in the second sample (upregulation) selected from the group of miR-505, miR-22, miR-30 (preferably miR-30e), miR-19 (preferably miR-19b), miR-378 (preferably miR-378a), and optionally also miR-20a and/or miR-146a,

and/or a lower level of at least one of the miRNAs in the second sample (downregulation) of miR-101.

Preferably, at least 2, at least 3 or at least 5 of the markers are upregulated.

In another embodiment of the invention, improving the fitness correlates with a higher level of at least one of the miRNAs in the second sample (upregulation) selected from the group of miR-19 (preferably miR-19b), miR-101 , miR-369 and miR-122,

and/or a lower level of at least one of the miRNAs in the second sample (downregulation) selected from the group of miR-30 (preferably miR-30e), miR-22, miR-505, miR-378 (preferably miR-378a), miR-494 and miR-135 (preferably miR-135a).

Preferably, at least 2, at least 3 or at least 5 of these markers are upregulated or downregulated.

In a preferred embodiment, at least one additional miRNA marker is selected from the group of miR-146, miR-20 and miR-10. It is preferred that the miR-146 is miR146-a, especially miR-146a-5p, and/or that the miR-20 is miR2-20a, especially miR-20a-5p. Preferably, the miR-146 and miR-20 are up-regulated in the method. Preferably, 2 or all markers from this group are selected. It is especially preferred that the additional miRNA markers are miR-146 and miR-20. Most preferably, at least the additional miRNA mR-20 is selected. It was found that changes of the levels of these markers can be significantly in the overall method. Thus, they can be used to complement and/or strengthen the results obtained with miRNA markers from the groups defined further above.

In a highly preferred embodiment, at least the three miRNAs miR-505, miR-22 and miR- 20, especially miR-20a, are selected.

In a preferred embodiment, the at least one miRNA marker is a total of 1 to 50 miRNA markers, preferably 5 to 30 miRNA markers or 8 to 25 miRNA markers, especially 10 to 50 miRNA markers, which are used in the method, and thus in steps (c) and (d). In methods with chips or the like, often the levels of several hundred miRNAs from a sample are analyzed in parallel. However, the inventive method is different from such known methods, because it comprises a purposeful analysis of markers which relate to the fitness of an individual in step (d). Therefore, in the inventive method it is sufficient to examine only a limited number of miRNAs from the sample. In the inventive method, it is not necessary, but also not detrimental to determine and compare the levels of other markers in step (c).

In a preferred embodiment, the difference of levels of at least one miRNA marker in the first and second sample has a P-value below 0.05, preferably below 0.03, more preferably below 0.01. In statistical analysis, the significance of a marker level change can be described by the P-value. In statistical hypothesis testing, it is generally assumed that a result is significant when the P-value is below 0.05. When it is found in the inventive method that P-value in such an order above can be attributed to the differences of miRNA marker levels, it can be concluded that the fitness of the individual has changed during the time period. Especially when the individual improved the lifestyle, the changes in the marker levels can indicate improvements.

In a preferred embodiment, the difference of levels of at least one miRNA marker is more than 1.0-fold, preferably more than 1.5-fold or more than 2.0-fold. Also for such changes, it may be concluded that the change of lifestyle during the time period between the first and second sample has improved the fitness.

In a preferred embodiment, at least one additional marker is analyzed by the method, which is not a miRNA. This can be advantageous, because different types of markers can provide complementary information about the fitness.

In a preferred embodiment, the at least one additional marker is a metabolite, protein or peptide, electrolyte, nucleic acid, carbohydrate or cell. Especially metabolic markers can provide additional valuable information regarding changes of fitness or health. In a preferred embodiment, the metabolite is a branched chain amino acid, bile acid, choline, beta-hydroxybutyrate or a ketone body.

In another preferred embodiment, the method is accompanied by an additional evaluation of physical conditions of the individual, such as heart beat, pulse, blood pressure, by electrocardiography or the like. Such non-invasive tests provide complementary information, which can support or confirm results obtained by the inventive miRNA marker analysis. When the method relates to evaluating and/or improving the fitness of the individual, it may comprise establishing a plan for achieving such purposes and/or providing it to the individual. Such a plan comprises one or more instructions for the individual how to change the lifestyle. In a preferred embodiment, improving the fitness of the individual in step (d) comprises establishing a plan for physical exercise, nutrition and/or lifestyle, and/or providing the plan to the individual. Only as an example, a plan for physical exercise may comprise directions for carrying out regularly moderate physical exercise. A plan for nutrition may comprise instructions for a diet, such as healthy nutrition or control of energy uptake. A plan for lifestyle may comprise combinations of various recommendations.

Subject of the invention is also a system for monitoring, evaluating and/or improving the fitness by the inventive method, comprising:

(A) Targets for at least two miRNA markers fixed to a support,

(B) means for determining the levels of at least one miRNA marker in the sample, (C) optionally means for obtaining, storing and/or handling the sample, and

(D) an instruction manual for carrying out the method.

The system is preferably a kit of parts. The at least one target for miRNA marker is attached to a support, such as a microarray, to which the miRNA which shall be determined can bind specifically. Means for determining marker levels in the sample could be appropriate solutions, chemicals or analytic devices. Means for obtaining, storing and/or handling the sample can be needles and devices for obtaining dried blood spots, microtubes or the like. The instruction manual is directed to the user who carries out the method. The system, especially in the form of a kit of parts, can be directed to laboratory staff, to skilled professionals or to the individual. Subject of the invention is also the use of at least one miRNA marker selected from the group of miR-505, miR-22, miR-30e, miR-19b, miR-101 , miR-378a, miR-494, miR-369, miR-122 and miR-135a for monitoring, evaluating and/or improving the fitness of an individual, wherein the use comprises determining the level of the at least one marker in a body fluid or fraction thereof obtained from the individual. In preferred embodiments of the invention, the markers and uses are defined as outlined above for the inventive method.

The inventive method, system and uses solve the problems defined above. A simple, reliable and precise method is provided for monitoring, evaluating and improving the fitness of an individual. It could be shown for a large group of individuals that some specific miRNAs are relevant markers for such purposes. A set of miRNAs was identified which is strongly correlated to fitness. The inventive method is relatively simple and convenient, since routine methods are available, especially based on microarrays, for determining levels of miRNA markers from large numbers of samples. Thereby, results are available within a short time, which are typically precise and reproducible. The inventive method can support individuals in improving their fitness by objective and relevant information. The method provides a reward to individuals who engage in such a challenge. Thus, the method can be a valuable tool for the individuals, but also for the professionals who assist them. FIG. 1 shows experimental results of levels of miRNAs of the participants in the study. Examples PILOT STUDY

A study was carried out to identify miRNAs which have a significant effect on the fitness of individuals. Preparatory work included more than 2 years of analysis of various miRNAs, epigenetic and metabolic markers in response to lifestyle, nutrition and aging processes such as inflammation. Also, interactions between a healthy, active lifestyle with metabolic aspects have been analyzed.

For the specific analysis of miRNAs, a pilot study was performed which is described in the following. A group of participants with controlled inclusion and exclusion criteria such as hormonal interactions were undergoing a tightly controlled training program. Training, nutrition and metabolic markers were analyzed at various time points. The expression changes of >200 miRNAs after onset of the training program was analyzed by Qiagen, DE, statistical analysis of correlations between mi RNA expression and training as well as correlation of additional lifestyle and nutrition factors was done performed in our group. In contrast to previous scientific studies of some miRNAs, a coherent set of > 200 miRNAs was analyzed in easy to handle dried blood spots (DBS) after a controlled training and nutrition plan, tightly monitored. miRNAs in our study showed often a specific expression profile. Additional markers and parameters of lifestyle and nutrition were included and analyzed to detect and exclude possible bias or important correlations. Results of the pilot project are outlined below.

Outline of methods and results of the pilot project analyzing changes of expression of >300 miRNAs in response to lifestyle changes towards a training program A study was carried out with individuals who performed a physical exercise program (sport intervention). miRNA markers were analyzed before and after the sport intervention.

Table 1 : Project workflow

Objective

The aim of this pilot study is to explore the effects of sport intervention on microRNAs in capillary blood. Participants

Participants in the study were 17 healthy middle-aged women with a slight overweight. The women had ages from 36 to 59 years and a BMI above 27 kg/m². Inclusion criteria were general health, normal weight or slight overweight, age between 35 and 60 years, no sport activities in the three to six months before the study. Exclusion criteria were normal or underweight, menopause, medication, diseases (such as high blood pressure, diabetes). Details about the participants are summarized in table 1. Three participants were excluded during the intervention (drop-out). Two participants took medication during the study (fluoxetine, orgametril). Two participants caught the flu during the spot intervention. As a consequence, the intervention was extended by the respective time period of one to two weeks.

Table 2: Participants

Physical exercise program

The participants underwent a fitness program which focused on strength and endurance and was carried out in a gym. The intensity of the sessions and the program was moderate. The program was carried out three to five times per week for 60 to 74 minutes. After four weeks, the program was increased to four times per week and 75 to 90 minutes. The total program was carried out for 10 weeks. In the first session in a gym, participants got an introduction to the training equipment and exercises to be carried out. In case participants could not carry out the program at the gym, exceptionally, they could complete a home workout plan. Before the intervention, participants complete a questionnaire about their diet, lifestyle and current athletic status. After completion of the intervention, an interview was conducted and the questionnaire was reviewed with the participants. During intervention, participants were controlled. They were contacted every two to three days by telephone, SMS and email, to ask them about the last training sessions and to give motivational tips if necessary. Further, the participants were accompanied every two weeks to the training to check if everything was carried out adequately.

Materials and Methods

Samples

The sampling was done on two time points: at the beginning of the study and at least 72 hours after the intervention. The participants were asked to fill a lifestyle and nutrition questionnaire and capillary blood was collected on protein saver cards (DBS; dried blood spots). The duration of the intervention was 10 weeks and included a fitness program which combined endurance and strength training. miRNA real-time qPCR

miRNA was extracted from the dried blood spots (DBS) with miRNeasy Micro Kit™ (Qiagen, DE). 30 ng RNA was reverse transcribed in 30 pi reactions using the miRCURY™ LNA (locked nucleic acid) RT Kit (QIAGEN). cDNA was diluted 100x and assayed in 10 mI PCR reactions according to the protocol for miRCURY™ LNA miRNA PCR; each miRNA was assayed once by qPCR on the miRNA Ready-to-Use™ PCR, Human panel I using miRCURY™ LNA SYBR Green master mix™. Negative controls excluding template from the reverse transcription reaction was performed and profiled like the samples. The amplification was performed in a LightCycler™ 480 Real-Time PCR System (Roche, CH) in 384 well plates. The amplification curves were analyzed using the Roche LC™ software, both for determination of Cq (by the 2^nd derivative method) and for melting curve analysis. The Cq value or cycle quantification value is the PCR cycle number at which the sample’s reaction curve intersects the threshold line.

Data analysis

The amplification efficiency was calculated using algorithms similar to the LinReg software. All assays were inspected for distinct melting curves and the Tm was checked to be within known specifications for the assay. Furthermore, assays must be detected with 5 Cq less than the negative control, and with Cq<37 to be included in the data analysis. Data that did not pass these criteria were omitted from any further analysis. Cq was calculated as the 2nd derivative. All data was normalized to the average of assays detected in all samples or alternatively to the average of custom defined assays detected in all samples.

Results

The miRCURY™ LNA miRNA PCR Human panel I includes 318 assays. On average, 219 miRNA were detected per sample and 160 miRNA were detected in all samples. Normalization is performed based on the average of the assays detected in all samples as this is shown to be the best normalization for qPCR studies involving numerous assays (Mestdagh et al. 2009).

Differentially expressed miRNAs post-sport vs pre-sport

When comparing the post-sport group to the pre-sport group using a t-test, 5 miRNAs were found to be differentially expressed using a cutoff of P-value < 0.05, as shown in table 3 below. The fold change was calculated when comparing post- versus pre-sport.

Table 3: Results

Differentially expressed miRNAs pre-sport vs post-sport

When comparing the pre-sport group to the post-sport group using a Paired t-test (with pairing factor Pair), 8 miRNAs were found to be differentially expressed using a cutoff of P-value < 0.05, as shown in table 4 below. The fold change was calculated when comparing pre- versus post-sport. The difference between the fold change to tables 3 and 4 is due to the different calculation of the values. Comparing pre- vs. post-sport and comparing post- vs. pre-sport results in positive or negative fold change. Table 4: Results

Differentially expressed miRNAs with biological relevance for physical activity in all 12 samples

When comparing the pre-sport group and the post-sport group using a Paired t-test, these 8 miRNAs were observed to be differentially expressed using a cutoff of P-value < 0.07, as shown in table 5 below. The fold change was calculated when comparing pre- versus post-sport.

Table 5: Results

The detailed results for each patient based are summarized in FIG. 1. Based on the analysis report of this study, microRNAs were chosen for tracking the influence of physical activity as molecular biomarkers as a part of an analysis panel. The summary also contains reported results from different tissues and settings. COMPREHENSIVE STUDY

A comprehensive study was carried out with a larger number of participants who underwent a 12 weeks physical exercise program. Selected miRNAs were analyzed in dried bloodspot samples. Unless noted otherwise, conditions, materials and methods were adjusted as described above. A flyer was designed to recruit the participants, which was launched in public places in Vienna and published on social media platforms. The target participants are inactive, have a BMI between 20 and 35 kg/m² and their age range is between 19 - 55 years (table 6).

Table 6: Study population

Bevor and after the intervention, the participants were required to complete a food frequency questionnaire (FFQ) and they had to undergo a bioimpedance analysis (BIA) to evaluate their nutrition, lifestyle and health status. The intervention phase lasted 12 weeks, whereby the participants had to increase the frequency and intensity of physical activities to 3-5 times a week, depending on their previous physical exertion. The training sessions were divided into two parts, endurance and strength training, both of which were trained in roughly equal amounts. One unit should be about 60 - 75 minutes. Furthermore, the participants were instructed not to change their diet for the duration of the study. In order to ensure adequacy of training, the participants trained under the supervision of the ISC fitness centre trainer. A weekly contact with the participants was maintained to ensure the motivation of the participants during the intervention and they had to keep a training diary, which was requested by the study staff once a week.

Dried bloodspot samples after the intervention were taken at least 72 hours after the last training session (T1). During this time, miRNAs which are affected by acute exercise can return to baseline levels. As typical for the calculation of mRNAs, 3 control (house keeping) miRNAs have been analysed and used for standardisation of miRNAs claimed as markers. The results are summarized in table 7.

In a control group of 15 volunteers with similar inclusion and exclusion criteria like the intervention group, miRNAs were analysed where questionnaires and at least 2 telephone interviews / week indicated no significant exercise or food additive intervention. No significant differences, changes or even trends were detected in blood spots comparing samples at time point 1 (TO) and a second time point 12 weeks later (T1) for the miRNAs selected as markers (P>0,5). The results for this control group demonstrate that long term changes of miRNA levels observed in the comprehensive study originate from the intervention.

The results show that the levels of miRNAs miR-19b, miR-20a, miR-22, miR-30e, miR- 101 , miR-146a and miR-378a, miR-505 underwent a long-term change due to the intervention. The results for miR-20a, miR-22 and mi-R505 are highly significant, because the p-values are far below 0.05. The results for other miRNA shown in the table, in combination with the previous results from the pilot study, suggest that they also underwent relevant long-term changes due to the intervention. Thus, the comprehensive study confirms that some specific miRNAs, which were identified in the pilot study above, undergo relevant changes after a long term sport intervention. The differences in p-values between the two studies can be explained by biological variances. The differences in the regulation (up/down) of miR-22, miR-505, miR-30e, miR-101 and miR-378a can most likely be explained by the temporal course of miRNA. Temporal effects on biomarker levels have been described in the art and can result from cycles of downregulation and re- expression. However, the data does demonstrate that there are long-term effects on the levels of the miRNAs, which have not been described in the literature before.

Table 7: Results of comprehensive study

A summary about findings from previous studies, if available, in which the markers have been described is provided in table 8. Interestingly, the present results are mostly different from other results described in the prior art, which relate to acute exercise. This demonstrates that the physiological foundation of fitness is different from short-time effects related to acute exercise, exhaustion and the like. Overall, the results from the pilot and comprehensive study demonstrate that some specific microRNAs can be used as significant, reliable and minimally invasive biomarkers for monitoring, evaluating and/or improving the fitness of an individual.

Table 8: Comparison to prior art

Literature:

Baggish et al. , "Dynamic regulation of circulating miRNA during acute exhaustive exercise and sustained aerobic exercise training", 2011 , J. Physiol. 16, 3893-3994.

Davidson et al., "High responders to resistance exercise training demonstrate differential regulation of skeletal muscle miRNA expression", 2011 , J. Appl. Physiol. 110, 309-317.

Domanska-Senderowska et al. “Expression analysis of selected classes of circulating exosomal miRNAs in soccer players as an indicator of adaptation to physical activity”, 2017, Biol Sport, 34(4), 331-338.

Fernandez-Sanjurjo et al.,“Circulating microRNA as regulators of the molecular response in exercise in healthy people”, 2016, Arch. Med. Deporte, 33(6), 394-403.

Fyfe et al.,“Concurrent exercise incorporating high-intensity interval or continuous training modulates mTORCI signaling and microRNA expression in human skeletal muscle”, Am J Physiol Regul Integr Comp Physiol., 2016, Jun 1 ;310(11), 1297-311

Keller et al.,“A transcriptional map of the impact of endurance exercise training on skeletal muscle phenotype”, J Appl Physiol (1985), 2011 , Jan; 110(1), 46-59.

Lee et al., "Biomarkers in sports and exercise: tracking health, performance and recovery in athletes", 2017, Journal of Strength and Conditioning Research, 31(10), 2920- 2937.

Margolis et al.,“Circulating MicroRNA Are Predictive of Aging and Acute Adaptive Response to Resistance Exercise in Men”, J Gerontol A Biol Sci Med Sci. , 2017, Oct 1 ; 72(10), 1319-1326.

Mestdagh, et al. “A novel and universal method for miRNA RT-qPCR data normalization”, 2009, Genome Biology, 10(6), R64.

Polakovicova et al., "Circulating microRNAs as potential biomarkers of exercise response", 2016, Int. J. Mol. Sci., 17, 1553.

Radom-Aizik et al.,“Evidence for microRNA involvement in exercise-associated neutrophil gene expression changes”, J. Appl. Physiol., 2010, 109, 252-261.

Radom-Aizik et al., “Impact of brief exercise on circulating monocyte gene and microRNA expression: Implications for atherosclerotic vascular disease”, Brain Behav. Immun., 2014, 39, 121-129.

Silva et al.,“MicroRNAs as Important Regulators of Exercise Adaptation”, Prog Cardiovasc Dis., 2017, Jun - Jul; 60(1), 130-151

Claims

1. A method for monitoring, evaluating and/or improving the fitness of an individual, comprising the steps of (a) providing a first sample, which is a body fluid or fraction thereof obtained from the individual at a first point in time,

(b) providing a second sample, which is the same body fluid or fraction thereof obtained from the individual at a second point in time,

(c) determining and comparing with each other the levels of at least one miRNA marker in the first and second sample, and

(d) monitoring, evaluating, and/or improving the fitness of the individual based on the result of the comparison made in step (c), wherein the at least one miRNA marker is selected from the group of miR-505, miR-22, miR-30, miR-19, miR-101 , miR-378, miR-494, miR-369, miR-122 and miR-135.

2. The method of claim 1 , wherein the individual changed his/her lifestyle between the first and second point in time.

3. The method of claim 2, wherein the change of lifestyle is expected to improve the fitness, such as a physical exercise program, a diet, a reduction of drugs and/or regular motion.

4. The method according to at least one of the preceding claims, wherein the time period between the first and second point in time is at least one week.

5. The method according to at least one of the preceding claims, wherein the method is also carried out with additional samples, which are obtained from the individual between the first and second point in time.

6. The method according to at least one of the preceding claims, wherein the body fluid is blood, saliva or urine, or wherein the samples are dried blood spots (DBS).

7. The method according to at least one of the preceding claims, wherein the miRNAs in the group are miR-505, miR-22, miR-30e, miR-19b, miR-101 , miR-378a, miR-494, miR-369, miR-122 and miR-135a.

8. The method according to at least one of the preceding claims, wherein at least one miRNA marker is selected from the group of miR-505, miR-22, miR-30, miR-19, miR-101 and miR-378.

9. The method according to at least one of the preceding claims, wherein at least 2 markers, preferably at least 3 markers or at least 5 markers are selected from the defined group of miRNAs.

10. The method according to at least one of the preceding claims, wherein at least miR-22 and miR-30e are selected from the group, and additionally miR-20, especially miR-20a is selected.

11. The method according to at least one of the preceding claims, wherein at least one additional miRNA marker is selected from the group of miR-146, miR-20 and miR-10.

12. The method according to at least one of the preceding claims, wherein at least one additional marker is analyzed by the method, which is not a miRNA, wherein the at least one additional marker is preferably a metabolite, protein or peptide, electrolyte, nucleic acid, carbohydrate or cell.

13. The method according to at least one of the preceding claims, wherein improving the fitness of the individual in step (d) comprises establishing a plan for physical exercise, nutrition and/or lifestyle, and/or providing the plan to the individual.

14. A system for monitoring, evaluating and/or improving the fitness by a method of at least one of the preceding claims, comprising:

(A) targets for at least two miRNA markers fixed to a support,

(D) an instruction manual.

15. The use of at least one miRNA marker selected from the group of miR-505, miR-22, miR-30e, miR-19b, miR-101 , miR-378a, miR-494, miR-369, miR-122 and miR-135a for monitoring, evaluating and/or improving the fitness of an individual, wherein the use comprises determining the level of the at least one marker in a body fluid or fraction thereof obtained from the individual.