Mitochondrial DNA Signatures of Poor Aerobic Exercise Trainability in Young Adults Born Preterm
Phase: NA
Conditions: Preterm Birth
Sponsor: Texas Tech University

Study Protocol & Statistical Analysis Plan 
Cover Page  
Study Title: 
Mitochondrial DNA Signatures of Poor Aerobic Exercise Trainability in Young Adults Born Preterm 
NCT Number: 
NCT06334107 
Document Date: 
December 11, 2025 
Protocol Version: 
v1.0 
Sponsor: 
American Heart Association 
Principal Investigator: 
Heather L. Vellers 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

STUDY PROTOCOL 
1. Specific Aims 
Specific Aim 1 (SA1): Compare maternal and VPTB young adult mitochondrial genomic 
characteristics and assess for unique mtDNA variants. 
Specific Aim 2 (SA2): Determine the effect of Aerobic Exercise Training (AET) on mitochondrial 
oxidative capacity and mtDNA heteroplasmy, copy number, and lesions in isolated PBMCs in VPTB 
and NTB young adults. 
2. Study Design and Procedures 
Specific Aim 1 Design and Procedures 
This aim involves collecting identifiable data, including subject weight, sex, race, and gestational 
age, from medical records. Blood will be collected via venipuncture from VPTB participants. Saliva 
samples will be collected from birth mothers, either via a mailed collection tube with instructions or 
during a facility visit. The birth mothers of the VPTB young adults serve as the control group for this 
aim. Ultra-deep sequencing of the full-length mitochondrial DNA (mtDNA) will be performed using an 
established laboratory and bioinformatic pipeline. The sequencing depth will be at least 1,000X, 
enabling accurate assessment of heteroplasmic sites and frequency. The assessment will identify de 
novo mtDNA variants (i.e., variants not present in the birth mother) in VPTB adults. 
Specific Aim 2 Design and Procedures 
This study will comprise a 16-week aerobic training intervention (walking/running) in young 
adults born with very preterm birth (VPTB ≤32 weeks) and normal-term birth (NTB). Age- and sex-
matched NTB young adults will serve as controls. Saliva and venous blood samples will be collected 
for PBMC isolation to compare mitochondrial genomic parameters (sequence, copy number, and 
heteroplasmy) at baseline and after the training intervention. 
Aerobic Exercise Training (AET) Program: The AET program will last 16 weeks, with sessions 
occurring 4–5 days per week for 40–60 minutes. The target intensity will be between 60–85% of the 
participant’s predetermined maximal heart rate (HR). To prevent injury and limit dropouts among 
sedentary participants, the frequency, duration, and intensity will slowly progress. 
• Week 1: Light intensity (60% HRmax) with flexibility for 4–5 days per week and 40–60 minutes 
duration. 
• Week 2: Intensity increases to 65–75% HR max. 
• After 8 weeks: Intensity increases to 75–85%, and the duration must be 60 minutes. 
• Subject progression (VO2max via graded exercise test) and exercise programming will be 
reassessed and altered at 4 and 8 weeks. 
Graded Treadmill Exercise Test: Participants in Aim 2 will complete a modified Balke maximal 
treadmill exercise test. After a 2-minute warmup at 2.5 mph, the speed will be adjusted to elicit 70–
75% of the age-predicted maximum heart rate (Tanaka equation: [208-(0.7xage)]). The incline will 
increase by 2.5% every 2 minutes until volitional fatigue. Maximal effort is established using three 
criteria: a heart rate within 10 bpm of the age-predicted maximum, a respiratory exchange ratio ≥1.1, 
and a rating of perceived exertion of at least 18 (using the 6-20 Borg scale). 
Laboratory Assessments: 
• Sample Collection: Blood samples will be collected via standard venipuncture, and PBMCs will 
be isolated via differential centrifugation. 
• Mitochondrial Function: Mitochondrial respiration in isolated PBMCs will be measured using 
the XFe24 Extracellular Flux Analyzer. PBMC Reactive Oxygen Species (ROS) production will be 
assessed via a fluorescent assay. 
• mtDNA Analysis (Aim 2): Heteroplasmy, lesions, and copy numbers will be assessed using 
PBMCs from VPTB and NTB participants. mtDNA lesions and copy numbers will be determined using 
a gene-specific quantitative PCR-based assay. 
3. Study Population and Recruitment 
Inclusion Criteria: 
• Age: 18–35 years old. 

• VPTB Group: Males and females born preterm with a gestational age of ≤32 weeks. Must be 
inactive (reported exercise < 150 minutes/week). 
• NTB Group (Controls): Age- and sex-matched subjects born at term (37 wks gestational age). 
Exclusion Criteria: 
• Diagnosed bronchopulmonary hyperplasia. 
• Diagnosed cardiovascular, metabolic, or renal disease, or signs or symptoms of these 
conditions. 
• Participants (and their birth mothers) with significant cardiometabolic events that might induce 
mtDNA variants are excluded. 
Recruitment: Recruitment will target individuals from Texas Tech University (TTU) and the 
Lubbock County community. Methods include email, local newspaper, radio, and social media 
advertisements. Informed consent will be obtained for VPTB adults to verify gestational age via 
medical records. Birth mothers will also be recruited to provide a saliva sample for mitochondrial 
genome comparisons. 
4. Sample Size (Power Analysis) 
A power analysis based on prior published data suggests a sample size of 25 subjects per 
group is needed to achieve 80% power using a two-way ANOVA with an alpha level of 0.05. To 
account for potential retention challenges, the study aims to recruit 30 subjects per group. This 
sample size is supported by other research that investigated mitochondrial respiration in VPTB young 
adults. 
 
 
 
STATISTICAL ANALYSIS PLAN (SAP) 
1. Results Analysis and Interpretation 
Specific Aim 1 Analysis 
The full-length mtDNA sequence will be compared between the VPTB young adult and their birth 
mother. Sites will be assessed for variants. A site is deemed an ‘informative’ mtDNA variant if it 
represents a change in amino acid sequence from the birth mother. 
• Expected Outcome (SA1): The study expects to identify informative mtDNA variants in VPTB 
young adults, likely in genes regulating oxidative phosphorylation subcomplexes and/or translating 
essential subunits of the respiratory chain complexes. 
Specific Aim 2 Analysis 
A Two-Way ANOVA will be employed to assess pre- to post-AET changes in key outcomes in 
VPTB and NTB young adults: 
1. PBMC mitochondrial respiration. 
2. mtDNA lesions and copies. 
3. VO2max. 
For AET-induced changes in the mean percent heteroplasmy frequency across heteroplasmic loci 
and the mean total number of heteroplasmic sites, a nonparametric statistical test such as the 
Wilcoxon rank test may be performed due to anticipated variability based on prior findings. 
Additionally, the study will assess differences in sites, genes, and regions where heteroplasmy is 
differentially affected by AET in VPTB and NTB young adults, specifically analyzing whether these 
align with a specific VPTB phenotype. 
• Expected Outcomes (SA2): 
    ◦ At baseline, VPTB adults are expected to have abnormally higher PBMC mitochondrial 
respiration compared to NTB adults. 
    ◦ Mitochondrial respiration is expected to decrease after AET in VPTB adults, suggesting a 
decline in function due to the exercise stressor overwhelming mitochondrial oxidant capacity. 
    ◦ AET is expected to decrease VPTB heteroplasmy frequency, while an increase is expected in 
NTB adults.