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Home , Epitestosterone

Search for additional biomarkers – multi-layered system

Human genome Blood doping E TranscriptomeTranscriptome x p o Exercise s Proteome o m e Gender

Influenced Metabolome Hypoxia Biomarkers Human systems

Progress report – 06.11.19 1 Transcriptomic biomarkers of blood doping: autologous blood transfusion

Blood Bone marrow Blood circulation Immature red blood cell manipulation

mRNA Protein Maturation

Erythropoiesis Red blood cell

Transfusion Clinical study

36 days of storage at 4°C

Blood Donation: ~500mL Blood Transfusion

-4 -1 +3h +6h +12h +1 +2 +3 +6 +9 +15

EDTA tubes Tempus RNA blood tubes

Progress report – 06.11.19 2 Confirmation using RT-qPCR and correlation with IRF

ALAS2 CA1 SLC4A1

RET% 2 1.5 1 0.5

RETICULOCYTES % RETICULOCYTES 0 -4 -2 0 2 4 6 8 10 12 14 16 Days

Progress report – 06.11.19 3 Outcomes 1

2016 2017

Blood transcriptome impacted by autologous blood transfusion

ALAS2, CA1, SLC4A1 genes as principal candidate markers

Variations more important than for conventional hematological parameters

Longitudinal monitoring of the number of transcripts may provide additional evidence of blood doping

Progress report – 06.11.19 4 Dried blood spots for antidoping purpose

Minimal invasiveness Direct detection of doping drugs Easy sampling

Increased frequency of sample collection Quantification of biomarkers Cost-effective transport

Progress report – 06.11.19 5 Quantification of ALAS2 RNA expression in dried blood spots

Sonication Transfer of DBS QIAzol Chloroform Centrifugation aqueous phase Elution excision

Incubation miRNeasy Mini kit 1. RNA extraction

Data treatmennt Amplification

ALAS2 Linear ALAS2 Linear + Circular 2. RT-qPCR

Progress report – 06.11.19 6 ALAS2 gene expression after blood withdrawal

Blood donation 16 healthy male volunteers 450 mL

-1 0 1 2 3 6 9 15 30 (days)

DBS EDTA Tempus RET% IRF% 2.5 18 16 * 2 * 14 12 1.5 10 8 1

Reticulocyte % 6

0.5 4 2 0 reticulocyte fractionImmature (%) 0 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 Time (days) Time (days)

Progress report – 06.11.19 7 ALAS2 gene expression after blood withdrawal

Blood donation 16 healthy male volunteers 450 mL

-1 0 1 2 3 6 9 15 30 (days)

DBS EDTA Tempus Tempus® Tube Dried blood spot 700 ** 800 *** ALAS2L ** ALAS2L 600 *** ** ** 700 ALAS2L+C ALAS2L+C 500 * 600 400 500 /Ref /Ref 400 300 ALAS2 *** ALAS2 300 200 ** ** 200 * 100 100

0 0 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 Time (day) Time (day)

Progress report – 06.11.19 8 ALAS2 gene expression after rhEPO injections

2 healthy male volunteers = 3’500 IU Eprex

-3 0 1 2 3 4 7 (days)

DBS Volunteer 1 Volunteer 2 120 120

100 100

80 80 /Ref /Ref 60 60 ALAS2 ALAS2 40 40 ALAS2L+C ALAS2L+C 20 20 ALAS2L ALAS2L

0 0 -3 -2 -1 0 1 2 3 4 5 6 7 8 -3 -2 -1 0 1 2 3 4 5 6 7 8 Time (day) Time (day)

Progress report – 06.11.19 9 Outcomes 2

DBS is a suitable matrix for gene expression analysis

No difference between venous and capillary blood for quantification of ALAS2 transcripts

Expression of ALAS2 gene is stimulated after blood withdrawal and rhEPO injections

Inclusion of ALAS2 circular form improves the magnitude of the response observed

Confirmation that ALAS2 is a promising candidate for the detection of blood doping through longitudinal monitoring

Progress report – 06.11.19 10 Biomarkers of doping - steroidomics

Human genome Blood doping E Transcriptome x p o Exercise s Proteome o Steroids m e Gender

Influenced MetabolomeMetabolome Hypoxia Biomarkers Human systems

Progress report – 06.11.19 11 Extended profiling of serum endogenous

Sample preparation Free steroids and Phase I metabolites Phase II metabolites 11-Deoxycorticosterone 11b-Hydroxyandrostenedione 5ααβ-Adiol, 17-Gluc -3-Sulf SPE 11-Deoxycortisol 11b-Hydroxyandrosterone 5ααβ-Adiol, 3-Gluc DHEA-3-Sulf OASIS HLB µElution (Waters) 17a-Hydroxyprogesterone 11b-Hydroxyepiandrosterone 5αββ-Adiol, 3-Gluc DHT-17-Sulf 21-Deoxycortisol 11-Dehydrocorticosterone 5βαβ-Adiol, 17-Gluc Epitestosterone-17-Sulf Loading 200 µL serum + 200µL H3PO4 4% 11-Ketoetiocholanolone 5ααβ-Adiol (5α-diol) 5βαβ-Adiol, 3-Gluc -3-Sulf Washing 400 µL H2O/MeOH 95/5 (v/v) + 0.1% NH4OH 16a-Hydroxyandrostenedione 5αββ-Adiol Androsterone-3-Gluc NorEtiocholanolone-Sulf Elution 50 µL MeOH/H2O 90/10 (v/v) 16a-Hydroxyprogesterone 5βαβ-Adiol (5β-diol) DHEA-3-Gluc Testosterone-17-Sulf Dilution 50 µl H2O 16a-Hydroxytestosterone 5βββ-Adiol DHT-17-Gluc -3-Sulf 16- EpiAndrosterone-3-Gluc -Sulf 18-Hydroxycorticosterone Androsterone 20a-Cortolone Epitestosterone-17-Gluc 5abb-diol-17-Sulf 20a- Etiocholanolone-3-Gluc 11-Ketoetiocholanolone-Sulf UHPLC-MS/MS analysis 20b-Cortolone NorAndrosterone-3-Gluc DHEA 3a,5a- NorEtiocholanolone-3-Gluc Kinetex C18 150 x 2,1 mm , ID 1.7 µm DHT 3a,5b-Tetrahydrocortisol Testosterone-17-Gluc Flow rate 300 µL/min Temperature 55°C Epitestosterone 3a,5b- 11-Ketoetiocholanolone-Gluc Etiocholanolone 7a-Hydroxytestosterone -3-Glu Phase A H2O + 5mM Ammonium Formate Phase B MeOH + 5mM Ammonium Formate Testosterone Pregnanediol Gradient 40% B to 80% B in 20 min

Progress report – 06.11.19 12 TestoFem Clinical study

Authorized by

Funded by

• 14 healthy women • No hormonal contraception • Regular menstrual cycles • No high-level sport

Study design: 3 phases = 3 menstrual cycles

Phase 1: Pre-treatment monitoring Phase 2: Testosterone gel treatment

Phase 3: Post-treatment monitoring

Progress report – 06.11.19 13 TestoFem Clinical study

So far

• 12 volunteers screened • 7 volunteers included • 2 volunteers completed the study • 1 volunteers completed treatment phase (phase III on-going) • 4 volunteers under treatment (phase II) • No adverse effect linked to the product reported • No drop-out • No treatment effect on the menstrual cycle

Progress report – 06.11.19 14 TestoFem Clinical study – Preliminary results steroid profile UHPLC-MS/MS

Testosterone

Testosterone Individual monitoring 5 8.0 7.0

V1 4 Volunteer 1 6.0 V2 5.0 3 4.0 V3 3.0 V4 Testosterone (nmol/L) Testosterone 2.0 2

Testosterone (nmol/L) Testosterone V5 1.0 V6

0.0 1 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 V7

Jours J+1 J+17 J+23 J+29 J+32 J+40 J+50 J+54 J+60 J+71 J+79 J+82

Jours

DHT

DHT 2.5 Volunteer 2 3.500

3.000 2.0 V1 2.500 V2

2.000 1.5 V3 1.500 V4 DHT DHT (nmol/L) DHT(nmol/L)

1.000 1.0 V5

0.500 V6

0.000 V7 0.5 0 10 20 30 40 50 60 70 80 90 Jours J+1 J+17 J+23 J+29 J+32 J+40 J+50 J+54 J+60 J+71 J+79 J+82

Jours

Progress report – 06.11.19 15 Outcomes of the clinical study

• Serum endogenous steroid profile (UHPLC-MS/MS analyses) • Urinary steroid profile (GC-MS analyses) • ‘Endocrinological’ profile - LH, FSH, SHBG, AMH, Ferritine, EPO (Immunology analyses) • Hematological profile (Sysmex XN-1000) • Targeted confirmatory GC-C-IRMS analyses • Untargeted steroidomic study for the discovery of potential biomarkers • Genotyping of UGT2B15 and UGT2B17 polymorphism • Body composition (DEXA scans) • Self-esteem (Rosenberg scale) and Quality of life (SF-36) • Transcriptomic study (Tempus tubes and/or DBS) • Evaluation of DBS as matrix for endogenous steroid profiling using UHPLC-MS/MS

Progress report – 06.11.19 16 Application of extended blood steroid profile to hyperandrogenic populations

Differentiation of populations with steroid profile Testosterone DHT 500 8000 400

50 6000 300 3646 DHT Testo 4000 200 100 2000

7 25 0 0

CAIS Neg PCOS CAIS Neg PCOS

Population Population

11b-OH-Androstenedione 16a-OH-Testosterone 400 25 4000 300 3000 200 X16aOHTesto 2000 X11bOHAdione 100 1000 0

CAIS Neg PCOS CAIS Neg PCOS Population Population Androsterone Sulf Etiocholanolone Sulf Androsterone Gluc Etiocholanolone Gluc

50 50 500000 39 5 24 140000 60000 5000000 28

400000 25 50000 4000000

100000 25 300000 40000 80000

3000000 5 AndroSulf EtioSulf EtioGluc AndroGluc

5 30000 60000 200000 1 2000000 20000 40000 5 100000 1000000 10000 20000 0 0 0 0

CAIS Neg PCOS CAIS Neg PCOS CAIS Neg PCOS CAIS Neg PCOS

Population Population Population Population

Progress report – 06.11.19 17 Perspectives

Inclusion of the 7 last volunteers for TestoFem clinical study Preliminary analyses of the samples collected during the clinical trial Validation of the quantitative UHPLC-MS/MS method Comparison of the blood steroid profile between hyperandrogenic populations and doped volunteers Comparison between urinary and blood steroid profile

Development of a workflow for the UHPLC-MS/MS analysis of DBS

Progress report – 06.11.19 18