Sperm DNA damage: The possible link between obesity and male infertility, an update of the current literature

Obesity prevalence worldwide is increasing significantly. Whilst maternal obesity has clear detrimental impacts on fertility, pregnancy and foetal outcomes, more recently there has been an increasing focus on the role of paternal obesity in human fertility. Recent meta‐analyses have indicated that obesity in men negatively affects basic sperm parameters such as sperm count, concentration and motility, increases the incidence of infertility and reduces the chances of conception. Sperm DNA damage, typically characterised by DNA strand breaks and oxidation of DNA nucleotides, is a specialised marker of sperm quality that has been independently associated with recurrent miscarriage, reduced assisted reproduction success and increased mutational loads in subsequent offspring. Whilst, there are still conflicting data in humans as to the association of obesity in men with sperm DNA damage, evidence from rodent models is clear, indicating that male obesity increases sperm DNA damage. Human data are often conflicting because of the large heterogeneity amongst studies, the use of body mass index as the indicator of obesity and the methods used for detection of sperm DNA damage. Furthermore, comorbidities of obesity (i.e., heat stress, adipokines, insulin resistance, changes in lipids, hypogonadism and obstructive sleep apnoea) are also independently associated with increased sperm DNA damage that is not always modified in men with obesity, and as such may provide a causative link to the discrepancies amongst human studies. In this review, we provide an update on the literature regarding the associations between obesity in men and fertility, basic sperm parameters and sperm DNA damage. We further discuss potential reasons for the discrepancies in the literature and outline possible direct and indirect mechanisms of increased sperm DNA damage resulting from obesity. Finally, we summarise intergenerational obesity through the paternal linage and how sperm DNA damage may contribute to the transmission.


INTRODUCTION
Obesity prevalence has tripled in the past 40 years worldwide. 1In Australia alone, two in three men are overweight (body mass index [BMI] >25 kg/m 2 ) or obese (BMI >30 kg/m 2 ). 2 Whilst, the detrimental effects of maternal obesity on pregnancy and foetal outcomes are clearly established, recent research indicates that paternal obesity also detrimentally affects fertility 3,4 and basic sperm parameters such as count, motility and morphology. 5,6[9] The data regarding the association between obesity and sperm DNA damage are currently, however, unresolved. 10This review will provide an update on the literature regarding the effect of obesity in men on fertility, basic sperm parameters and sperm DNA damage, comment on why there may be large discrepancies in the literature as to the effect of obesity in men on sperm DNA damage, and outline the implications of obesity on the health of the next generation with insights on how DNA damage may contribute to the transmission.

UPDATE ON THE EFFECTS OF OBESITY ON MALE FERTILITY
2][13][14][15][16] As of 2017, the major conclusion of these metaanalyses indicated that male obesity was associated with multiple changes in basic sperm parameters, namely reductions in sperm count, concentration and semen volume. 13Rates of infertility were also increased in men with obesity, 11 although the impact of male obesity on assisted reproduction outcomes was inconclusive. 11,14Updated metaanalyses published between 2020 and 2022 (assessing publications up until 2019) consistently show negative associations with reduced semen volume and sperm count, although they report differing outcomes in terms of sperm concentration, viability and morphology. 5,6,16ce again, obesity was found to be associated with higher rates of infertility, 12 although no clear difference in assisted reproduction outcomes was identified. 15The following section will review the relevant articles published in the past 5 years to provide an update on the relationship between obesity, pregnancy outcomes and basic sperm parameters.

Pregnancy and assisted reproduction
A few studies have assessed the role of paternal obesity on pregnancy outcomes in the previous 5 years (Table 1).8][19] Three of these studies utilised men with clearly documented infertility, 4,17,18 and found higher BMI in the infertile cohort in two of the three studies. 4,17Another study utilised self-reported infertility (based on survey questions regarding prior infertility treatment) and did not identify a difference in BMI between the fertile and infertile groups. 19o further studies used time to pregnancy cut-offs to determine subfertility.One study from China of 50,927 couples utilised a cut-off of 6 months to determine subfertility and identified no relationship to paternal BMI. 20In contrast, a Norwegian study of 28,341 women and 26,252 men utilised a cut-off of 12 months' time-to-pregnancy to determine subfertility and found that both maternal and paternal obesity was associated with longer times-to-pregnancy. 3They also identified that the genetic risk score for obesity was also associated with higher rates of infertility.2][23] Two were performed in the general population, and identified that maternal obesity had a much greater effect on birth outcomes, whilst paternal overweight/obesity had minor if not nil effect. 22,23In contrast, the final study assessed a cohort of couples with recurrent pregnancy loss, identifying poorer fecundability outcomes with BMI ≥30 kg/m 2 . 215][26][27][28][29][30] In all cases bar one, 24 BMI was the only method of adiposity classification utilised (using race-based norms).8][29][30] Three studies also assessed the effects of obesity in men on fertilisation rates, with two 24,27 of three 29 studies showing negative associations with obesity in men.

Basic sperm parameters
Numerous cohort studies published in the previous 5 years have assessed the relationship between BMI and basic sperm parameters (volume, concentration, count, motility and morphology) (Table 2).
Notably, despite these studies originating from many locations across the world, including China, Shanghai, India, Argentina, Turkey, Iran, Brazil, Madrid, Canada, Egypt, France, Taiwan, Ukraine, Budapest and America, there is a significant bias in that 70% (21/30) solely focus on men of couples with infertility or subfertility.The total sample size of these studies varied significantly.Over half (16/30) of these studies had a sample size less than 200 men, whilst only nine studies had a sample size greater than 1000 (six of which were in infertile populations).It is possible that some of the smaller studies lacked sufficient power to detect minor changes in parameters associated with obesity.When assessing only the nine studies with populations greater than 1000 men, obesity was more consistently associated with decreased sperm concentration (7/9 studies), decreased motility (8/9 studies) and decreased normal morphology (5/6 studies).The most recent meta-analysis assessing the relationship between paternal obesity and basic sperm parameters consisted of 28 relevant articles published between 2004 and 2019, and identified very similar findings to these larger studies of the previous 5 years, namely, that increased BMI was significantly associated with reduced semen volume, sperm count, concentration and viability. 5

THE EFFECT OF OBESITY ON SPERM DNA DAMAGE
8][9] A two-step hypothesis has been theorised for the origins of sperm DNA damage in human spermatozoa. 61The first step involves disruption to spermiogenesis that generates defective spermatozoa with vulnerable chromatin, and the second step involves the attack of this vulnerable chromatin by reactive oxygen species (ROS), which leads to the formation of oxidised DNA adducts and DNA strand breaks placing the spermatozoa in a state of oxidative stress. 61The latest systematic review and meta-analysis understanding the relationship between obesity in men and sperm DNA damage identified no association between sperm DNA damage in men with obesity (BMI >30 kg/m 2 ) and men of normal weight (BMI 18.5-24.99kg/m 2 ) (Standard Mean Difference = 0.85; confidence interval [CI]: −0.16, 1.87, p = 0.10). 10e majory limitation of current studies assessing sperm DNA damage and obesity are the plethora of methods used to determine whether or not sperm DNA damage has occurred. 62,63Direct measurement of sperm DNA damage can occur through the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, which fluoresces in proportion to dUTP binding to strand breakages.
Sperm DNA damage can also be indirectly measured through tests such as the sperm chromatin structure assay (SCSA), which utilises acridine orange to identify strand breaks in denatured DNA, the chromatin dispersion test, which assesses the formation of 'halos' (dispersed DNA) after denaturation (a process impaired by DNA fragmentation) or the Comet assay, by which degree of DNA damage is proportional to the 'comet' appearance of denatured DNA under electrophoresis.Whilst results between tests do correlate, each requires differing thresholds to determine clinical significance. 64The current meta-analysis assessed 14 studies (10 cross-sectional and four cohort with a total of 8255 men) utilising only SCSA, TUNEL and sperm chromatin dispersion because of the calculation of a DNA fragmentation index.
The variability in measures and differing cut-offs may account for inconsistent results of the meta-analysis, including finding significant increases in sperm DNA damage with certain subgroups of obesity (BMI 30-34.9kg/m 2 ) compared to normal weight men (BMI <25 kg/m 2 ) but not with all obese men (BMI >30 kg/m 2 ). 10 Due to this latest meta-analysis, we only assessed additional articles published from 2018 onwards.

Humans
Cohort studies in humans published in 2018-2022 also show conflicting results, matching findings of the recent systematic review and meta-analysis (Table 3).Three out of the eight studies (37%) showed no effect of men with obesity on sperm DNA damage, whilst four out of the eight studies (50%) showed that male obesity and/or increasing BMI was associated with increased sperm DNA damage (Table 3).Additionally, the last study that assessed the effects of obesity on couples experiencing repeat pregnancy loss found that sperm DNA damage showed no correlation with the prognosis for a live birth, despite men with obesity having a lower chance of achieving a live birth. 21

Animal models
Given the large heterogeneity amongst human studies, animal models can be more informative for understanding the true effects obesity has on sperm DNA damage.Rodent models utilising high-fat diets (commonly high in saturated fats ranging 20%-40%) mimicking a [72][73] Comorbidities associated with obesity have also been shown to significantly amplify the effects of obesity on sperm DNA damage.
For instance, obese diabetic mice 74 and obese mice not supplemented with vitamin D 72 showed a greater propensity for sperm DNA damage, whilst leptin-deficient obese mice showed impaired testicular structure, which may increase the propensity for sperm susceptibility to DNA damage through the male reproductive tract.

WHY THERE MAY BE DESCREPANCIES AS TO THE EFFECT OF OBESITY ON SPERM DNA DAMAGE
As outlined above, the current evidence determining whether obesity in men is associated with increased sperm DNA damage is not consistent in humans.Notably, the most common measurement of adiposity when assessing outcomes was BMI, which has significant inter-racial variation and limitations in differentiating between lean and fat mass. 76Indeed, other measures such as the visceral adiposity index or waist circumference may correlate better with adiposity-associated complications. 24,77Furthermore, evidence from rodent models suggests that there may be an interplay with obesity-related comorbidities and sperm DNA damage.This appears to be replicated in humans, as there is very clear evidence that metabolic syndrome, a condition characterised by obesity and associated complications such as hypertension, dyslipidaemia and/or insulin resistance, is associated with significant impairment in sperm count, motility, morphology and increased sperm DNA damage. 78 such, variability in human studies is likely because of the presence/absence of comorbidities that exacerbate sperm DNA damage in those men with obesity.The following section will expand on this notion.

Direct mechanisms of increased sperm DNA damage resultant from increased adiposity
There are multiple methods by which increased adipose tissue can directly induce DNA damage, including secreted hormones, disruption of the hypothalamic-pituitary-gonadal (HPG) axis and direct impacts on heat regulation of testes (Figure 1).

Leptin
Adipose tissue is a hormonally active tissue that secretes a vast array of hormones known as adipokines, including adiponectin, ghrelin, leptin, orexin, obestatin and various other inflammatory cytokines. 79e disruption of these hormones in obesity accounts for many of the complications of obesity.A predominant hormone of obesity is leptin, the concentration of which correlates directly to degree of adiposity. 80Whilst all its effects are not yet confirmed, there are clear implications for regulating food intake, body weight, the hypothalamicpituitary-thyroid and hypothalamic-pituitary-growth hormone axes, bone formation and inflammation. 813][84] Whilst similar studies have not been performed in humans, leptin receptors are present on germ cells and Leydig cells of human testis, and expression is increased in infertile populations, suggesting a similar mechanism. 85,86

Inflammation
Further adipokines such as tumour necrosis factor, interleukins, leptin and resistin induce a systemic inflammatory response, characterised by macrophage infiltration of adipose tissue proportional to degree of adiposity. 87Systemic inflammation induced by obesity results in multiple changes throughout the reproductive tract.Rodent models have shown that obesity induces increased ROS formation and reduced germ cell number in the testis. 88,89There is also disruption of Sertoli cell function contributing to reduced spermatogenesis. 90yond the testis, there are increased inflammatory markers in the epididymis, seminal vesicles and prostate glands, 91 with corresponding increased seminal plasma ROS. 92Similar findings of increased seminal ROS formation and inflammatory markers have been identified in men with obesity. 93,94As such, obesity appears to induce an inflammatory effect throughout the entire reproductive tract that correlates with increased ROS formation, creating a hostile environment for sperm formation and growth.Whilst no studies have directly assessed the relationship between degree of inflammation and sperm DNA damage, there is clear evidence in other cell types that oxidative stress induces DNA damage, 95 and as such a similar mechanism likely contributes to poorer sperm quality in men with obesity. 96

Hypogonadism
A concurrent complication of increasing adiposity is functional hypogonadism, which occurs through multiple different mechanisms, including aromatisation of testosterone to oestrogen 97 and leptin-related suppression of the HPG axis. 98,99In rodent studies, testosterone has been found to modulate the anti-oxidant system of spermatozoa, 100 and as such obesity-related hypogonadism could exacerbate ROS-related sperm dysfunction.Hypogonadism also pre-disposes men to increasing insulin resistance (below) and further weight gain, 101,102 perpetuating a cycle of deteriorating sperm quality and increased sperm DNA damage.

Heat
Physical changes around the testes themselves may also be implicated in increasing sperm DNA damage.Rodent studies show that transient increases in scrotal temperature induce spermatocyte DNA damage and are associated with reduced pregnancy rates. 103In these mice, heat stress directly induced degeneration of germ cells; however, apop- in those with obesity compared with normal weight men 104 and may account for some of the changes in basic sperm parameters such as count and motility.

Indirect mechanisms of increased sperm DNA damage associated with increased adiposity
Additionally, many changes that occur more commonly in people with obesity can also exacerbate DNA damage.The most likely contributing factors in this case include insulin resistance/diabetes mellitus, changes in lipid metabolism and obstructive sleep apnoea (OSA) (Figure 2).

Insulin resistance
Insulin resistance is characterised by the inability of cells to respond to normal levels of insulin, requiring supra-physiological concentrations to maintain normoglycaemia, whereas type 2 diabetes mellitus occurs when, despite supraphysiological secretion, insulin concentrations are inadequate with resultant hyperglycaemia. 105,106esity is a major risk factor for diabetes mellitus because of the effects of adipokines and inflammation, yet not all people with obesity develop diabetes.The degree of insulin resistance is moderated by multiple factors, including fatty acid concentrations, gut microbiome and diet. 105,107,108Currently, the International Diabetes Foundation estimates that approximately 10.6% of the world population (aged 20-79 years) have impaired glucose tolerance, and 10.5% have diabetes mellitus. 109Rates of obesity and diabetes have increased significantly worldwide in the past decades, 1,110 and without significant intervention, the prevalence can only increase.
2][113] Sperm DNA damage in type II diabetes is likely mediated through increased ROS formation within the testes and epididymis, 114 although the exact mechanism is currently undetermined.In somatic cells, established pathways of ROS formation (and subsequently DNA damage) include protein kinase C-dependent activation of NADPH oxidase, alteration of the NADH/NAD+ ratio resulting in 'pseudohypoxic' conditions and advanced glycation end product (AGE) formation. 115Of these, AGEs have been most extensively studied in spermatozoa.AGEs are glycosylated proteins that typically occur in states of hyperglycaemia.They can directly damage and impair the function of proteins, but also bind to the AGE receptor (RAGE) to activate multiple intracellular pathways that result in increased ROS formation, inflammation and apoptosis. 116Current studies of diabetic men show increased AGE concentration of their seminal plasma, 117,118 and upregulation of RAGE on spermatozoa, 119,120 with sperm RAGE concentrations positively correlating with the degree of sperm DNA damage. 119 a rodent model, RAGE upregulation corresponds with increased C-Jun-N terminal kinase activity in spermatocytes, indicative of apoptosis induction. 121Similarly, mitigation of hyperglycaemia also results in reduced mitogen-activated protein kinase activity, 122,123 a pathway identified to be activated by RAGE in somatic cells. 124In vitro, incubation of spermatozoa with AGE-forming molecules in both F I G U R E 2 Mechanisms of sperm DNA damage generation with obesity-related comorbidities.Baseline mechanisms of DNA damage induced by obesity outlined in Figure 1 shown (brown).Additive methods of DNA damage can occur from diet, which is associated with reduced Ω-3 fatty acid consumption and is associated with increased insulin resistance.Ω-3 fatty acids are associated with increased anti-oxidant capacity; therefore, reduced consumption lowers the anti-oxidant capacity of spermatozoa.Similarly, increased insulin resistance may result in hyperglycaemia with associated increased advanced glycation end product production and reactive oxygen species (ROS) formation.Finally, obstructive sleep apnoea, a common condition associated with increasing adiposity, results in intermittent hypoxia, which has been shown to increase ROS (animal models).This exacerbates the disparity between ROS formation and anti-oxidant capacity resulting in increased sperm DNA damage.Theoretically, addressing these additive changes (green) would result in reduced sperm DNA damage, yet further studies are required to confirm their benefits.
non-diabetic rodents and humans is associated with reduced sperm motility, mitochondrial activity and fertilisation rates and increased sperm DNA damage. 125,126though type II diabetes is clearly associated with increased sperm DNA damage, there is a significant knowledge gap as to the effect of insulin-resistant obesity.

Lipid metabolism
Lipid products are necessary for appropriate sperm formation.They are a component of the cellular membrane of spermatozoa, but are also a source of energy for Sertoli cells, and alter gene expression in these cells based on availability. 129In obesity, lipid metabolism is often deranged and characterised by elevated triglycerides and free fatty acids because of increased release from adipocytes and poor clearance. 129Increased free fatty acids disrupt insulin function and exacerbate insulin resistance, 130,131 further adding to detrimental effects outlined above.
The relative concentrations of fatty acids have significant implications for sperm structure and function.Polyunsaturated fatty acids are essential fatty acids obtained from diet and form up to 50% of total fatty acids in mammalian spermatozoa. 1293][134] There are many proposed mechanisms by which lipid constitution affects sperm quality, including altering sperm formation, anti-apoptotic effects of Ω-3 fatty acids and altering intracellular enzyme activity. 135There is also a clear association between Ω-3 fatty acids and oxidative stress, as shown by improvement in antioxidant markers of seminal plasma 136 and markers of sperm DNA damage 137,138 with Ω-3 fatty acid supplementation in men.Sperm docosahexaenoic acid and palmitic acid (Ω-3 fatty acids) have also been found to be decreased in overweight and obese men, with the degree of deficiency correlating to amount of DNA damage present. 139 these fatty acids are unable to be synthesised by the body, their effects are soley due to dietary habits.The 'Western diet' , well known to be associated with obesity, has been shown to have a greater proportion of Ω-6 fatty acids, 140 and raises the possibility that dietary change irrespective of weight loss may moderate DNA damage in this cohort. 141

Obstructive sleep apnoea
OSA is a condition characterised by intermittent obstruction of the airway during sleep.It is a significantly underdiagnosed condition affecting up to one in three men, with greater prevalence with increasing age and obesity. 142Obesity predisposes to OSA through changes in airway mechanics because of increased adiposity, reduced lung volumes because of visceral fat deposition, loss of ventilation stimulation by leptin (leptin resistance) and somnogenic effects of pro-inflammatory cytokines. 143,144,145 human studies have assessed sperm DNA damage in OSA; however, a rodent model of intermittent hypoxia mimicking OSA identified increased testicular oxidative stress, impairment in sperm motility and reduced pregnancy rates following intermittent hypoxia. 146The exact mechanism is currently unknown; however, intermittent arterial hypoxia is associated with intermittent testicular hypoxia and likely downregulates anti-oxidant capacity of germ cells.8][149] Given the association between ROS and DNA damage, 95 it is likely that OSA-induced oxidative stress, either directly through intermittent hypoxia or indirectly through exacerbated inflammation and insulin resistance, will result in increased DNA damage.
OSA is also associated with reduction in serum testosterone concentration, with the most recent meta-analysis indicating that this change is beyond the effect of underlying obesity. 150Cur-rently, there is limited evidence that continuous positive airway pressure (CPAP) therapy results in improvement in serum testosterone concentrations, 151 although this meta-analysis assessed only 388 men with obesity-related comorbidities, and appropriate use of

TRANSGENERATION PATERNAL OBESITY-THE ROLE OF SPERM OXIDATIVE DAMAGE
Despite the negative effects obesity in men has on their reproductive capabilities, it is now known that these detrimental effects are transgenerational.It is estimated that 40 million children under the age of five years and more than 330 million children and adolescents aged 5-19 years are overweight or obese. 152Obese children are five times more likely to be obese in adulthood, representing a lifelong social and economic burden. 153A major risk factor for childhood obesity is paternal obesity. 154Cohort studies indicate that obesity in fathers is associated with (i) increased child BMI between 1 and 22 years of age, (ii) increased fat mass of 3-month-old babies and (iii) higher weight growth velocities and BMI of children into adulthood. 126][157][158][159][160][161][162] Evidence from rodent models indicates that this effect is mediated by factors other than a common living environment, 163 which points to components delivered by spermatozoa at conception.Lane et al. 164 provided some of the first evidence that elevated ROS and associated DNA damage in spermatozoa are specifically involved as a cause of transmission of obesity to offspring.Through in vitro exposure of mouse spermatozoa to H 2 O 2 to elevate ROS concentrations prior to IVF, they were able to recapitulated many offspring metabolic phenotypes induced by rodent models of paternal obesity. 164The exact mechanism by which obesity-associated increased ROS and by-products in spermatozoa contribute to offspring obesity and metabolic syndrome is yet to be determined.One potential hypothesis, found in other paternal pathologies is through increased transmission of de novo mutations caused by oxidised DNA at fertilisation.For instance, advanced paternal age increases spermatozoa de novo mutational rate by approximately two additional mutations per year of life.This is the dominant factor in determining the de novo mutational rate inherited by offspring. 165Whilst on an individual level the effect is low, the de novo mutational load induced by delayed paternity of mid-late 40s is predicted to increase offspring risk of brain-related disorders (i.e., schizophrenia and intellectual disability) by 9%-20%. 166ese findings indicate that paternal obesity-induced ROS changes are likely promutagenic (Figure 3).Elevated ROS concentration in spermatozoa can cause DNA strand breaks and oxidation of DNA nucleotides proportional to intracellular ROS. 167,168Oxidative damage to DNA can induce several sugar and base modifications, single and double strand breaks, nucleobase modifications as well as covalent crosslinks. 169The most commonly observed lesion on DNA is 8-hydroxy-2′-deoxyguanosine (8OHdG), where the guanosine base is modified by the hydroxyl radical (OH), a free radical that can damage all basic biomolecules. 170,1713][174] Specifically, at least in humans, it was found that there are selected areas of the sperm genome that are prime targets for oxidative attack (i.e., chromosomes 3, 5, 8, 9, 15 and 18) and that these are not random. 174As increased sperm oxidative stress is not only limited to male obesity, targeted attacks to the male gamete may help explain some of the similarities in offspring phenotypes seen across many male reproductive pathologies. 175erm histone retention (incomplete protamination) is a common feature seen in overweight and obese men. 25 Significantly, the regions that retain histones (H2A, H2B, H3 and H4) in sperm DNA are conserved amongst mammalian species, indicating their significance for successful embryo formation. 176The DNA regions that are associated with retained histones are not randomly distributed and appear to be specifically enriched at loci of developmental importance, including signal factors, HOX gene clusters, transcription factors, microRNA clusters and imprinting gene regions (which carry a parent of origin allele molecular memory generated early in the germline). 177Our preliminary findings show that 8OHdG lesions from obesity co-localise (but not exclusively) with histone H3 in both mouse and human spermatozoa (Figure 3).The localisation of 8OHdG to histone and nuclear matrix-bound regions in spermatozoa from male obesity is significant given that bound histone segments are not remodelled by the oocyte post-fertilisation and are some of the first areas to be transcribed in the paternal genome. 178,179As such, increased histone retention and 8OHdG DNA lesions in spermatozoa have the potential to sig-nificantly modify early embryonic events resulting in a cascade of aberrant downstream offspring phenotypes 176 and programming of altered metabolic health (Figure 4).Furthermore, evidence for this comes from a previous study showing that high-fat diet-fed rodents have enriched histone H3 retention in spermatozoa at genes important for embryogenesis (including embryo morphogenesis), transcription and DNA binding factors 162 and as such these areas are likely more venerable to oxidative attack.
Interestingly, spermatozoa can only partially repair 8OHdG lesions and only contain the first enzyme in the BER pathway (OCG1), which is able to remove the oxidised base from the DNA, creating an abasic site and relies on the remaining enzymes (APE1 and XRCC1) to be delivered by the oocyte at fertilisation to complete its repair. 180If the early embryo (zygote) is unable to repair 8OHdG delivered by spermatozoa prior to the first round of paternal genome replication (occurring in the PN3-PN5 zygote), this can give rise to an 8OHdG mismatch, commonly because the 8OHdG is seen as a T and not a G, resulting in a G:C → T:A transversion mutation. 1818OHdG has a second chance of being repaired after the first round of DNA replication by DNA glycosylase, APE1 and DNA polymerase, which can insert the correct C opposite the 8OHdG base.However, other kinds of DNA mutations are also possible including deamination of cytosine to uracil 181 as well as indirect loss of methyl marks. 171,182Increased DNA damage repair of the paternal genome post-fertilisation is morphologically seen as a delayed first and second cleavage event, occurring because of late paternal genome replication. 183Coincidently, in both humans and rodent models, male obesity is associated with failed fertilisation and delayed first, second and third cleavage events as well as compaction and blastocyst formation [184][185][186][187][188][189] and as such hints at a similar mechanism.Sperm 8OHdG repair in the early embryo is not always complete, and can be inherited by offspring, with unresolved 8OHdG lesions in gametes created by triple knockout mice (TOY-KO) showing 37 times higher somatic cell mutation rate in their offspring. 190We have also shown that 8OHdG lesions from male mice fed a high-fat diet persist in the late zygote, 191 although they manifest at a reduced incidence compared with spermatozoa, suggesting that some repair may have occurred.Furthermore, in an under nutrition mouse model, sperm 8OHdG lesions were moderately negatively correlated with offspring pre-pubertal postnatal weight (d14 −0.424 and d21 −0.374, p < 0.01) independent of founder diet. 192Of particular interest, the presence of 8OHdG in DNA from other tissues has been associated with a number of age-related diseases including Alzheimer's disease, Parkinson's disease and amytrophic lateral sclerosis (in neurological acute encephalopathy) 193 and may help explain offspring phenotypes described as a result of paternal obesity.This is because mutations formed post-fertilisation can contribute to somatic cell instability 194 and may play a part in the development of polygenic obesity (the most common form of obesity) (Figure 4). 195cently, it has been found that RNA may be more vulnerable to ROS than any other cellular component. 196Sperm harbour a unique RNA population that encapsulates both non-coding and protein-coding RNAs that are delivered to the oocyte at fertilisation, 197 with small non-coding RNAs (sncRNAs, <200 nt) implicated in the transmission Five-week-old C57BL6 male mice were allocated to either a high-fat diet (SF00-219, Specialty Feeds) or a control diet (SF04-057, Specialty Feeds) for a period of 10 weeks to induce obesity. 156Spermatozoa were collected from the vas deferens and epididymis and incubated in G-IVF PLUS (Vitrolife) medium.Human semen was collected from either normal weight or obese men undergoing assisted reproductive treatment.Motile sperm populations were isolated following a swim up in G-IVF PLUS.Spermatozoa from both species were fixed onto poly-L-lysine slides in 4% paraformaldehyde.8OHdG staining of spermatozoa was performed by a modified protocol of 173  of obesity from fathers. 198Interestingly, similar enzymes used in the repair of 8OHdG are also involved in the degradation of oxidised RNAs, with oxidised sncRNA dysregulating their targets. 199This suggests that sncRNAs oxidised in spermatozoa because of obesity could be either degraded prior to or at fertilisation, and thus either fail to act upon their cognate targets or switch their targets through mispairing (Figure 4).The antibodies that pick up oxidative DNA (8OHdG) are also specific for oxidised RNA lesions, indicating that increased oxidative damage in spermatozoa because of obesity also generates oxidised RNA (Figure 3).More recently, oxidised RNAs have been shown to be signalling modulators responding to cellular oxidative stress and have been related to the pathogenesis of a number of human diseases including neurodegenerative, metabolic and cardiovascular conditions, and cancer, 196 also hinting at their ability to potentally modify offspring phenotypes.Further evidence is required to determine if the oxidised RNA profile of spermatozoa from males who are obese is even altered and what the subsequent effects on early embryogenesis and offspring phenotypes are.

CONCLUSION
The was incomplete, resulting in formation of spermatocytes with increased DNA damage.In humans, scrotal temperatures are elevated F I G U R E 1 Direct mechanisms for how increased adiposity can cause sperm DNA damage.Increased adiposity can cause DNA damage through multiple direct mechanisms.Leptin directly damages spermatozoa in animal models, as does exposure to increased testicular temperature (which occurs in obesity because of increased adiposity).Pro-inflammatory adipokines induce an inflammatory state that induces reactive oxygen species (ROS) formation in seminal fluid.Finally, disruption of the hypothalamic-pituitary-gonadal (HPG) axis results in decreased testosterone concentrations, which have been shown to modulate anti-oxidant capacity (in animal models).The net effect is increased ROS formation with reduced anti-oxidant capacity, resulting in increased sperm DNA damage.
CPAP was not assessed.Further studies are required to assess sperm quality, including DNA damage in BMI-and comorbidity-matched men with or without sleep apnoea, and whether appropriate use of CPAP alleviates the multiple detrimental effects on the reproductive system.As shown, there are multiple mechanisms by which obesity can induce sperm DNA damage; however, many of these mechanisms are not solely because of increased adipose tissue and therefore cannot be captured by BMI, which is by far the most prevalent method for classification of obesity in human cohort studies.Concurrent comorbidities and lifestyle factors such as insulin resistance, diabetes, OSA and diet quality also likely contribute to DNA damage formation and as such need to be assessed in conjunction with conventional methods of obesity classifications (i.e., BMI) in studies moving forward.Whilst, further research is required to confirm their role and determine appropriate time of intervention, they outline how addressing comorbidities of an obese individual may be more important than focusing on BMI for optimising semen quality.Furthermore, it provides possible methods for improving fertility outcomes (e.g., dietary change, insulin sensitising medications, CPAP) in addition to weight loss.
role of paternal obesity in fertility and birth outcomes is currently an area of increasing knowledge and research.Recent studies clearly show that obesity is associated with changes in basic sperm parameters such as sperm concentration and motility.Whilst fertility outcomes are not as clear, there is evidence of longer time to pregnancy and poorer assisted reproduction outcomes in obese men.Animal studies clearly F I G U R E 4 Inheritance of oxidative lesions from spermatozoa at conception in the intergenerational transmission of obesity via fathers.High concentrations of reactive oxygen species (ROS) in spermatozoa from obesity increase (1) oxidative DNA and (2) RNA lesions to spermatozoa that are inherited at conception, reducing pronuclear formation and embryo growth and increasing the inheritance of de novo mutations that increase obesity and metabolic disease risk to subsequent offspring.showthat obesity is associated with increased sperm DNA damage, and whilst human studies are not as consistent, variations in participant ethnicity, inadequacy of obesity measurement tools (body mass index), variation in DNA damage measurement and lack of assessment of concurrent confounders (comorbidities) likely account for this variation.The presence of increased sperm DNA damage has implications not only for pregnancy outcomes but also for intergenerational effects, predisposing to an obesity phenotype in future generations.Further research is required to delineate the exact mechanisms of increased sperm DNA damage from obesity to identify means of preventing a continuous cycle of worsening obesity intergenerationally worldwide.

Study Population Sample size Obesity (BMI) relationship to sperm volume Obesity (BMI) relationship to concentration (million/mL) Obesity (BMI) relationship to sperm count (mil- lion/ejaculate) Obesity (BMI) relationship to motility Obesity (BMI) relationship to morphology Incorporated into recent meta- analysis
An update on the literature (2017-2022) for the effect of obesity in men on basic sperm parameters.
the rest identified no clear association.Fifteen of 30 (50%) studies identified a decrease in sperm concentration, whilst nine of 17 (53%) identified decreases in total sperm count.The most consistent finding was that of decreasing motility, identified in 70% (21/30) of studies.Only 10 of 22 (45%) studies assessing morphology identified a negative association with increasing BMI.TA B L E 1 An update on the literature (2017-2022) for the effect of obesity in men on pregnancy and assisted reproduction outcomes.WC (<90 cm, 90.1-100.5 cm, >100.5 cm) BMI (18.5-25 kg/m 2 , >25 kg/m 2 ) 75

value Definition of obesity Measure of sperm DNA damage Main outcomes
TA B L E 3 An update on the literature (2018-2022) for the effect of obesity in men on sperm DNA damage.Abbreviations: BMI, body mass index; CI, confidence interval; IVF, in vitro fertilisation; SCSA, sperm chromatin structure analysis; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; WC, waist circumference; WHR, waist circumference over hip circumference; WHtR, waist circumference over height; 8OHdG, 8-hydroxy-2′-deoxyguanosine.As such, evidence from rodents is clear that obesity increases sperm DNA damage when excessive adiposity is induced by poor nutrition.