A man referred to an infertility clinic is assessed first by semen analysis, performed according to the laboratory manual of the World Health Organization, now in its sixth edition (WHO, 2021). The test counts spermatozoa and grades their motility and morphology. It was never designed to examine the DNA inside the sperm head, and men with entirely normal results may still father no pregnancy. Interest in sperm chromatin grew from that observation and from the early demonstration by Evenson et al. (1980) that the susceptibility of sperm DNA to denaturation differed between fertile and subfertile males.

Sperm DNA fragmentation, usually abbreviated SDF, refers to single-strand and double-strand breaks in the nuclear DNA of the spermatozoon. Meta-analyses have associated higher fragmentation with miscarriage (Robinson et al., 2012) and with poorer results after in vitro fertilisation and intracytoplasmic sperm injection (Simon et al., 2017). Clinicians who order the test are therefore soon asked a practical question by the couple in front of them: whether the result can be improved. The usual answer is advice about smoking, drinking, weight, heat and supplements.

That advice is reasonable on general health grounds. Whether it is supported by evidence specific to sperm DNA is a separate matter, and it is the subject of this review. The focus is on men attending infertility clinics, because that is where the test is used and where most of the data come from, and because findings in such men cannot be assumed to hold in the general population or the reverse.

This is a narrative review of the published literature. It draws on landmark laboratory papers, on meta-analyses and large observational studies where they exist, and on the practice guidelines of professional societies. No formal search protocol was applied and no pooled estimates are offered. The aim is to set out what is reasonably established, what is suggested but unproven, and where the literature is too heterogeneous to support a firm statement. Occupational and iatrogenic causes of DNA damage, such as chemotherapy and radiotherapy, lie outside the scope, as does the technical question of sperm selection for assisted reproduction.

Four assays dominate clinical use. The sperm chromatin structure assay (SCSA) uses flow cytometry and acridine orange to measure the proportion of cells whose DNA denatures after a mild acid challenge, reported as the DNA fragmentation index (Evenson et al., 1999). The TUNEL assay labels the free 3'-hydroxyl ends of broken strands with a terminal transferase and counts the labelled cells by microscopy or flow cytometry. The sperm chromatin dispersion test infers fragmentation from the absence of a halo of dispersed DNA loops after denaturation and protein removal (Fernández et al., 2003). The comet assay, in alkaline or neutral form, measures DNA migration from single cells during electrophoresis.

These tests do not measure the same thing. TUNEL and the comet assay detect breaks directly, while SCSA and the dispersion test detect susceptibility to denaturation, which is related to breaks but also to chromatin compaction. Thresholds differ by assay and by laboratory. A fragmentation index of about 30 percent in the SCSA has been widely used to mark a group with reduced chances of natural and intrauterine conception (Evenson et al., 1999; Bungum et al., 2007), but that figure does not transfer to other methods. Reviews of the tests have stressed this lack of standardisation for many years (Sakkas and Alvarez, 2010).

The consequence for the present question is direct. A study that reports higher fragmentation in smokers using one assay and a study that finds no difference using another are not necessarily in conflict, and results cannot simply be averaged. Professional guidance reflects the uncertainty. The American Society for Reproductive Medicine concluded that the evidence did not support routine use of the test (ASRM, 2013), and the joint guideline of the American Urological Association and the same society does not recommend it in the initial evaluation of the infertile couple (Schlegel et al., 2021). Other expert groups have set out specific scenarios, including unexplained infertility, recurrent pregnancy loss, varicocele and lifestyle risk, in which they consider testing informative (Agarwal et al., 2016; Esteves et al., 2021).

The mature spermatozoon is unusually exposed. Its cytoplasm, and with it most of its antioxidant enzymes, is shed during spermiogenesis; its membranes are rich in polyunsaturated fatty acids; and it has very limited capacity to repair DNA. Protection depends on the tight packaging of the genome around protamines and on the antioxidants of seminal plasma. Aitken and De Iuliis (2010) proposed a two-step account in which faulty chromatin remodelling during spermiogenesis leaves some cells poorly protaminated, and these vulnerable cells are then attacked by reactive oxygen species. Other recognised routes include abortive apoptosis in the testis and damage acquired during transit through the epididymis (Sakkas and Alvarez, 2010).

Oxidative stress is the mechanism through which most lifestyle exposures are thought to act. Reactive oxygen species arise from leukocytes in semen, from immature spermatozoa that retain excess cytoplasm and from systemic sources, and their production is increased by smoking, obesity, heat and pollutants (Tremellen, 2008; Wright et al., 2014). Figure 1 summarises this common pathway. The model is coherent and supported by laboratory work, but coherence of mechanism is not evidence of effect in patients, and the sections that follow consider each exposure against the clinical data.

Table 1 summarises the exposures most often discussed with patients, the mechanism proposed for each and the general state of the evidence. The paragraphs below give the reasoning behind each entry.

An association between an exposure and DNA damage does not show that removing the exposure repairs it. The strongest test of the oxidative model in practice has been antioxidant supplementation. The Cochrane review of antioxidants for male subfertility found that supplements might increase live birth rates, but judged the evidence to be of low certainty because the trials were small and at risk of bias (Smits et al., 2019). The subsequent multicentre randomised trial by Steiner et al. (2020) found that a combined antioxidant preparation did not improve semen parameters or DNA integrity in men with male factor infertility. For smoking cessation, alcohol reduction and exercise, comparable trials with sperm DNA as an outcome are not available.

Two further cautions follow. Spermatogenesis takes roughly ten weeks, so any intervention must be sustained before its effect can be assessed, and single measurements are subject to considerable biological variation within the same man. In addition, the outcome that matters to couples is a healthy child. A fall in fragmentation index is a surrogate, and its relation to live birth after lifestyle change has not been established. Advice to stop smoking, moderate alcohol and lose excess weight can be given with confidence on general health grounds; the claim that it will correct a DNA fragmentation result should be made with more reserve.

Several weaknesses recur across this literature. Most studies are cross-sectional and recruit from clinics, where men with risk factors and men with poor results are both over-represented. Exposure is self-reported. Assays and thresholds vary, and few studies adjust for age, abstinence, varicocele and genital tract infection together. Publication of positive associations is more likely than publication of null results. Each of these problems can be addressed: by prospective cohorts with repeated sampling, by agreed reporting of assay method and abstinence, by objective markers such as cotinine for tobacco exposure, and by trials of structured lifestyle programmes that follow couples to live birth.

There is also a question of who should be tested. If lifestyle counselling is appropriate for all men seeking fertility care, a test is useful only where its result changes management, for example by identifying men for whom varicocele repair, a shorter abstinence interval or a different mode of assisted reproduction should be discussed. That is the logic of the scenario-based recommendations (Agarwal et al., 2016), and it deserves prospective evaluation.

Sperm DNA fragmentation is higher, on average, in men who smoke, who are obese and who are older, and a shared oxidative mechanism makes these associations credible. For alcohol, heat and environmental pollutants the evidence is suggestive and incomplete. The literature is weakened throughout by cross-sectional design, by confounding within clinic populations and by assays that do not agree with one another. Whether changing behaviour lowers fragmentation, and whether a lower value brings a better chance of a child, are questions that current studies do not settle. Clinicians can encourage healthier habits while being candid about what the test can and cannot promise.

To be declared by the author(s) before publication.

To be declared by the author(s) before publication.