Introduction
Male factor infertility contributes substantially to infertility, but conventional semen analysis does not fully assess sperm function. Sperm DNA fragmentation (SDF), damage to the genetic material in sperm cells, has been proposed as a marker of male reproductive potential. However, SDF testing is not currently part of routine fertility assessment in Denmark, and the prevalence and clinical relevance of elevated SDF among Danish men undergoing fertility treatment are not well established.
Oxidative stress and lifestyle-related factors may contribute to sperm DNA damage. Lifestyle modification may therefore be a potential approach to lowering SDF. However, evidence from randomized controlled trials evaluating the effect of a comprehensive lifestyle intervention on SDF is limited.
The overall aim of this prospective research programme is to investigate the prevalence and clinical significance of SDF among men undergoing fertility treatment and to evaluate whether a 3-month individualized lifestyle intervention can reduce SDF and improve subsequent reproductive outcomes.
Study design and study components
The study is a prospective research programme consisting of three integrated components:
- A cross-sectional study investigating the prevalence and extent of SDF among men undergoing fertility treatment.
- A prospective cohort study investigating the association between SDF levels and subsequent fertility treatment outcomes.
- An embedded parallel-group, superiority randomized controlled trial evaluating a 3-month individualized lifestyle intervention in men with elevated SDF.
Study workflow
The three study components are integrated within the same clinical workflow. All participants initially undergo SDF assessment. The SDF result determines whether a participant is eligible to enter the randomized controlled trial.
After providing written informed consent, participants provide a semen sample for SDF assessment and routine semen analysis and a blood sample for assessment of hormonal and other health markers. The semen sample is already part of routine fertility care, although participation in the study may require an additional visit to the clinic. Participants with an SDF of 20% or lower remain in the prospective cohort and continue their fertility treatment according to routine clinical practice. Participants with SDF above 20% are eligible for randomization.
Randomized controlled trial
Participants eligible for the randomized controlled trial are randomly assigned in a 1:1 ratio to either the individualized lifestyle intervention or standard fertility care. Randomization is performed using a computer-generated block randomization sequence with variable block sizes. Allocation is managed through REDCap, and the randomization sequence is maintained by an independent third party to preserve allocation concealment.
Because of the nature of the lifestyle intervention, participants cannot be blinded to treatment allocation. However, laboratory personnel assessing post-intervention SDF and study physicians assessing fertility treatment outcomes remain blinded to allocation. Samples and study data are pseudonymised using unique study identification numbers.
Lifestyle intervention
The intervention lasts for 3 months and is individualized according to information collected through a baseline self-assessment questionnaire. The intervention addresses several potentially modifiable factors associated with sperm quality and oxidative stress including diet, physical activity, smoking and nicotine use, alcohol consumption, sleep, stress management and vitamin and antioxidant supplementation. Participants have access to a study-specific lifestyle mobile application designed to support adherence to the intervention. The application functions as a brief daily diary in which participants record diet, physical activity, sleep and stress. A follow-up telephone call is conducted halfway through the intervention period to support adherence, address questions and encourage continued participation.
Control group
Participants randomized to the control group receive standard fertility care according to routine clinical practice at the clinic and do not receive additional lifestyle counselling as part of the study. A usual-care comparator was chosen because no evidence-based lifestyle intervention specifically targeting SDF is currently part of standard fertility care. This allows the study to assess the additional effect of the individualized lifestyle intervention while maintaining standard clinical management for participants in both groups.
Biological samples and laboratory assessments
Routine semen analysis is performed at the fertility clinic in accordance with the applicable World Health Organization recommendations. A portion of the semen sample is retained for SDF assessment and is divided into aliquots for analysis and cryopreservation. Total SDF, including both single- and double-stranded DNA breaks, is assessed using the Lens Hooke R10 system based on the Sperm Chromatin Dispersion Assay. Double-stranded DNA breaks are assessed using the Lens Hooke R11 system based on the Sperm DNA Fragmentation Releasing Assay. Prior to study testing, analytical validation is performed to assess the reliability and reproducibility of the systems. Pre-analytical conditions, including abstinence period, liquefaction time, sample temperature and time from collection to analysis, are standardised and documented in standard operating procedures. Blood samples are collected at baseline from all participants. Blood samples are analysed for cholesterol, glucose, vitamin D and relevant reproductive hormones.
Lifestyle questionnaires and follow-up
Participants in the randomized controlled trial complete an electronic baseline self-assessment questionnaire covering lifestyle factors including diet, exercise, smoking, alcohol, caffeine intake, stress and dietary supplements. A follow-up questionnaire is completed after 3 months by participants in both the intervention and control groups. In the intervention group, the questionnaire is used to assess adherence to the recommended lifestyle changes and to complement information recorded in the lifestyle application. In the control group, it is used to assess whether participants independently changed relevant lifestyle behaviours during the study period. After the 3-month intervention period, participants in the randomized controlled trial provide a new semen sample for SDF testing and a second blood sample and complete the follow-up questionnaire. Fertility treatment outcomes are obtained from electronic medical records.
Statistical analysis
The study is powered primarily to detect a difference in SDF levels between the intervention and control groups. The sample size for the randomized controlled trial is based on data from a previous pilot study conducted by the research group, in which a mean reduction in SDF of 7.2% was observed. Assuming a standard deviation of 10%, corresponding to an estimated Cohen's d of 0.72, 64 participants are required to provide 80% power to detect a statistically significant between-group difference using a two-sided alpha level of 0.05. Allowing for an anticipated 20% dropout rate, 80 participants will be recruited for the randomized controlled trial. Approximately 20% of participants in the cohort are expected to have SDF above 20%. Assuming that approximately half of the eligible participants invited to the randomized controlled trial will participate, approximately 800 men will therefore be recruited to the cohort to ensure sufficient recruitment for the randomized trial. Statistical analyses will be performed using Stata. For the randomized controlled trial, the primary analysis will follow the intention-to-treat principle and compare post-intervention SDF levels between the intervention and control groups. Between-group differences in continuous outcomes will be assessed using linear regression or analysis of covariance, with adjustment for baseline SDF where appropriate. Binary outcomes, including live birth and early pregnancy loss, will be analysed using logistic regression. Per-protocol analyses will be conducted as sensitivity analyses. The extent and pattern of missing data will be evaluated, and appropriate statistical methods will be applied where necessary.
Ethical considerations and risks
The study involves minimal additional risk. Semen collection is part of routine fertility care, although participation may require an additional clinic visit. For participants randomized to the lifestyle intervention, fertility treatment is temporarily postponed during the 3-month intervention period. The postponement is considered ethically acceptable because it is clinically justified by the biological duration of spermatogenesis and may potentially improve the likelihood of successful fertility treatment following the intervention. The possibility of postponement is explicitly explained to participants before consent to the randomized controlled trial is obtained. Participants may decline participation in the randomized controlled trial and continue with fertility treatment according to the original treatment plan without consequences for their clinical care. Participants randomized to the control group continue standard fertility treatment without postponement. Blood sampling may cause mild discomfort. Potential psychological discomfort related to lifestyle assessment and feedback is recognised. Counselling will therefore be provided in a respectful and supportive manner. The lifestyle intervention is considered low risk, and serious adverse events are not anticipated. Unexpected adverse events or unintended consequences will be recorded and managed according to applicable regulatory requirements.
The study is expected to provide information that may improve understanding of the role of SDF in male infertility and determine whether a non-invasive lifestyle intervention can modify SDF. At an individual level, the intervention may improve male reproductive health and potentially increase the likelihood of successful fertility treatment. More broadly, the findings may contribute to future assessment of the role of SDF testing and lifestyle-based approaches in fertility care.
