Omega 6/Omega 3 Ratio Is High in Individuals with Increased Sperm DNA fragmentation

An imbalance between omega-6 and omega-3 fatty acids in sperm has been linked with lipid peroxidation and DNA damage in sperm, indicating a possible correlation to fertility potential. This cross-sectional study involved 56 infertile men (aged 25–45), and assessed the relationship between the omega-6 to omega-3 fatty acid ratio in sperm and seminal plasma with sperm DNA fragmentation. Individuals were categorized based on high or low levels of sperm DNA fragmentation according to two tests (TUNEL and SCSA assay less or greater than 10 and 30%, respectively), and their fatty acid composition, as well as sperm functional tests, were analyzed. Results showed that men with high DNA fragmentation exhibited higher percentages of total saturated, monounsaturated, and omega-6 to omega-3 fatty acid ratios in both sperm (P < 0.001) and seminal plasma (P < 0.001) compared to men with low DNA fragmentation. The percentage of sperm lipid peroxidation, and residual histone (P < 0.05) were higher, while the percentage of sperm motility (P < 0.001) was lower in the former compared to the latter group. Moreover, Pearson's correlation revealed positive associations between the omega-6 to omega-3 fatty acid ratio with sperm lipid peroxidation, DNA fragmentation, and residual histones in both sperm and seminal plasma. Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better sperm quality compared to men with a lower omega-3 fatty acid.


Introduction
Several studies have suggested that sperm membrane fatty acid composition may influence the functional characteristics of spermatozoa [1,2].Polyunsaturated fatty acids (PUFAs) are essential constituents of all human cells [3], involved in maintaining the properties of the plasma membrane lipid bilayer, thus, play a vital role in the fertility status [4].However, due to the lack of delta-5 and delta-6-desaturase enzymes, the human body is not capable of synthesizing the essential PUFAs de novo; thus, it is necessary to acquire them through food consumption [5].Being present at a high concentration in human spermatozoa, omega-3 PUFAs have been linked to the integrity and fluidity of the sperm plasma membrane [6] and successful fertilization [7].It's worth mentioning that docosahexaenoic acid (DHA) exists at extreme levels in human ejaculates [8][9][10].Lower concentration of DHA in seminal plasma and spermatozoa has been clearly linked to the increased mean melting point, and decreased fluidity of the spermatozoa membrane, resulting in poor semen quality and fertility status [9][10][11][12].This is related to both escalated oxidative stress and decreased levels of a-linolenic acid intake, which is the precursor of DHA [9] in seminal plasma.
According to former studies, about 30 to 80% of male subfertility cases might be due to the destructive effects of oxidative stress on spermatozoa.This could be caused by the harmful effect of ROS by inducing lipid peroxidation and DNA damage [13,14].Induced oxidative stress in seminal plasma is a consequence of the imbalance between reactive oxygen species (ROS) generation and the body's scavenging capacity [15].In addition, spermatozoa are highly vulnerable to oxidative damage since they possess a limited amount of cytoplasm [9,12].Extreme spermatozoa DNA damage can significantly impair male fertility [16][17][18], and has been linked to lower acrosome reaction, birth defects, and childhood cancer in the offspring [19].Furthermore, it has been recognized that oxidative stress to sperm DNA results in elevated DNA fragmentation [20][21][22].The intensity of these damages can be measured by the sperm terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) as well as sperm chromatin structural assay [23] following a microscopic or flow cytometry analysis.
To prevent the destructive effects of ROS on DNA fragmentation, several exogenous anti-oxidants have been supplemented in men's diet to support the sperm anti-oxidant systems which scavenge the ROS, resulting in reduced internal cellular damage [24].It is well-documented that omega-3 PUFAs possess great anti-oxidant properties [24,25].Due to their ease of accessibility and exceptional safety profile, many studies have used omega-3 PUFAs as a nutraceutical product in order to improve semen quality in humans [24][25][26] and different species of domestic animals [27][28][29].In a former study conducted by Safarinejad et al., the fatty acid profile of fertile and infertile men diagnosed with idiopathic oligo-asthenic-teratozoospermia was evaluated and compared with each other.The authors found that fertile men had higher spermatozoa levels of omega-3 PUFAs, as compared with their infertile counterparts.Moreover, infertile men had a higher ratio of serum omega-6 to omega-3 fatty acids, when compared to fertile men [2].In another study, Attaman et al. evaluated the relationship between semen quality and dietary fats in 99 men who attended a fertility clinic and revealed that higher dietary intake of omega-3 PUFAs was positively correlated with sperm morphology [30].
In addition, other pieces of literature have indicated that increased omega-6 to omega-3 fatty acids ratio in spermatozoa has also been involved in poor semen quality in oligozoospermic and/or asthenozoospermic men [8].This means that a diet containing different proportions of omega-3 or omega-6 fatty acids can alter PUFAs in sperm membranes [31].In this sense, for the first time, the current study aimed to assess the relationship between sperm and seminal plasma omega-6 to omega-3 fatty acids ratio with sperm classic parameters, lipid peroxidation, and DNA damage in two populations of men attended the fertility clinic diagnosed with low and high sperm DNA damage according to TUNEL assay results as well as sperm chromatin structure assay (SCSA) results.

Study Participants and Semen Collection
This cross-sectional study was conducted using semen samples of men seeking infertility treatment at Isfahan Fertility and Infertility Center (IFIC), between September 2021 and May 2022.The range of men's age was between 25 and 45 years.Among participants who were referred to this center, daily between 2 to 4 cases were included in this study.A part of the semen sample was immediately separated for the assessment of sperm DNA fragmentation tests (SCSA & TUNEL assays).On the rest of the sample, half of the remaining semen sample entered the process of separating sperm from seminal plasma, and then the separated sperm and seminal plasma were frozen, so that their fatty acid profile could be examined in the future.The other half of the semen sample was considered for the assessment of sperm parameters and functional tests.Semen samples that had TUNEL and SCSA values of lower or higher than 10 and 30%, respectively [32] were included in the study (70 samples in total).A total of 4 and 10 samples were excluded from the study due to their fatty acid profile and low semen volume, respectively.Ultimately, 56 samples which consisted of 28 semen samples with a high degree of sperm DNA damage value and 28 semen samples with a low degree of sperm DNA damage value (SCSA & TUNEL) entered the study.Further, fresh semen samples were centrifuged following the separation of seminal plasma from sperm pellet for assessment of fatty acid composition.Then, the sperm pellet was resuspended and washed with phosphate-buffered saline (PBS) and then divided into two parts; one part for assessment of fatty acid composition, and another for evaluation of sperm functional tests.

Ethical Approval
All participants signed and wrote the informed consent form, and agreed to participate in this study after its aim was explained to them.Semen samples were collected via masturbation into sterile plastic containers, after 3-5 days of sexual abstinence according to World Health Organization (2010) criteria [33].

Sperm Concentration
Semen volume was calculated using the weighting method.For assessment of sperm concentration, semen samples were diluted in 1% formalin, and then in sodium bicarbonate solution (1:10) and placed on a Counting Chamber Neubauer (Marienfeld, Germany).Applying an optical microscope (LABOMED CxL; 20 ×), samples were assessed by a trained laboratory technician and the observations were expressed as million sperms per milliliter.For sperm count, sperm concentration was multiplied by semen volume and expressed as a million per ejaculation.

Sperm Motility
The sperm motility was assessed utilizing computerassisted sperm analysis, following LABOMED CxL optical microscope.Ten microliters of semen were placed in a pre-warmed sperm counting chamber and the chamber was covered with a coverslip.For each sample, at least five different microscopic fields were assessed.According to World Health Organization (2010), four distinct types of sperm motility including rapid-progressive, slow-progressive, non-progressive, and immotile were counted.Ultimately the percentage of total sperm motility for each sample was reported [33].

Abnormal Sperm Morphology
Sperm morphology was assessed by Diff-Quick staining according to World Health Organization (2010) [33].A smear was prepared for each sample stained with eosinophilic xanthene and basophilic thiazine solutions.Abnormalities in the head, neck, and tail regions were counted under high microscopic magnification (× 1000), and the result was expressed as a percentage of sperm abnormal morphology.

Sperm DNA Damage by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling
Briefly, a washed semen aliquot containing 2-3 × 10 6 sperm was equally divided into negative and positive control and test tubes.Then, the sperm pellet was fixed in 4% paraformaldehyde for 30 min and washed with PBS.Permeabilization of sperm was carried out with 0.2% Triton X-100 in PBS for 5 min, followed by the addition of 50 µl of the staining solution (TUNEL kit; Apoptosis Detection System Fluorescein, Promega, Mannheim, Germany) to negative and positive control test tubes for 1 h (37 °C; dark room).For the positive control tube, the samples were incubated with DNase I (40 IU/ml for 10 min) to induce DNA damage before the fixation step, while for the negative control tube, no enzyme was added.Ultimately, samples were washed two times with PBS and analyzed by flow cytometry (FACScan BD FACS Calibur, Becton-Dickinson, San Jose, CA, USA).For each tube, at least 10,000 sperm were assessed [23].

Sperm Chromatin Structure Assay
Two million sperms were separated from the fresh sample, one million for the negative control tube, and one million for the test tube.Then, the volume of each tube was raised to 1 ml by a TNE + NaCl + EDTA buffer.For the test tube, 400 μl acid-detergent solution was added to 200 μl of the diluted semen sample within 30 s, while for the negative control tube, no acid-detergent solution was added.Then, both mixture tubes were stained with 1200 μl of acridine orange (Sigma, St. Louis, USA) staining solution.At least 10,000 sperm were analyzed per tube using a flow cytometer (FACSCalibur Becton Dickinson, San Jose, CA, USA), and the results were expressed as a percentage of sperm DNA fragmentation index [16].

Sperm Residual Histones or Immature Chromatin Packaging
Briefly, for each washed sample, one smear was prepared and fixed with 3% glutaraldehyde.Then, the slides were stained with 5% aqueous aniline blue in a 4% acetic acid solution.The slides were then dehydrated in successive ethanol baths (70, 96, and 100%) and exposed to xylol solution for 5 min.In the next step, the dehydrated slides were covered with Entelane and at least 200 sperms were evaluated under high microscopic magnification (× 1000).Sperm with a dark blue nucleus were considered to have immature chromatin packaging [23].

Fatty Acid Composition in Seminal Plasma and Sperm
The fatty acid content of seminal plasma and spermatozoa was analyzed using High-performance liquid chromatography [34].Briefly, aliquots of 300 µL seminal plasma were transferred into glass tubes for direct transesterification.To each sample, 2 mL of methanol-benzene (4:1, v/v) was added along with the addition of an internal standard (heptadecanoic acid, C17:0) and 0.01% butylhydroxytoluene as an anti-oxidant.Samples were vortexed at low speed while slowly adding 200 µL of acetyl chloride, within 2 min.Samples were then heated for 60 min at 100 °C in a heating block and shaken continuously.Then, 5 mL of 6% (w/v) potassium carbonate solution was added to the cooled tubes at room temperature.The samples were vortexed for 30 s and centrifuged at 900 g for 20 min at 15 °C.The fatty acid methyl esters (FAMEs) accumulated in the upper benzene phase were transferred to gas chromatography vials and stored at 4 °C until chromatograph evaluation.In the case of spermatozoa, 2-10 × 10 6 cells were diluted with one volume of PBS and centrifuged at 900 g for 8 min.The resulting pellet was resuspended in 400 µL of PBS.Then aliquots of 300 µL of sperm suspension were treated in the way previously explained by Martínez-Soto et al. [10].

Statistical Analysis
Data normal distribution was tested by Shapiro Wilks normality test.The significance of differences was assessed by Student's t-test and the values were expressed as mean ± SD.The potential relationships between measured parameters were explored using Pearson's correlation test.All analyses presented in this study were conducted using SPSS software (version 26.0, Chicago, IL, USA), with a significance level of P < 0.05.

Comparison of Sperm and Seminal Plasma Fatty Acid Composition
The participants' fatty acid composition of sperm and seminal plasma, including the percentage of saturated, monounsaturated, polyunsaturated, omega-6, and omega-3 fatty acids, are detailed in Table 2.
The mean values of total saturated, and monounsaturated fatty acids were markedly higher (P < 0.001) in both sperm (53% and 27% increase, respectively) and the seminal plasma (12% and 11% increase, respectively) of men with a high degree of sperm DNA damage, as compared that of those with a low degree of sperm DNA damage.Similarly, the amount of total omega-6 fatty acids significantly elevated in both sperm and the seminal plasma by 22% and 25% respectively, in men with a high degree of sperm DNA damage, as compared that of those with a low degree of sperm DNA damage.The mean values of total omega-3 fatty acids and total polyunsaturated fatty acids were markedly lower (P < 0.001) in spermatozoa of men with a high degree of sperm DNA damage, as expected.In addition, the ratio of omega-6 to omega-3 fatty acids was significantly promoted in both sperm (a 2.5-fold increase, P < 0.001) and the seminal plasma (80% increase, P < 0.001) of men with a high degree of sperm DNA damage as compared to men with a low degree of sperm DNA damage.

Comparison of Sperm Parameters and Sperm Functional Tests
Among numerous semen samples assessed daily for SCSA and TUNEL assay, 28 samples with high sperm DNA fragmentation and 28 samples with low sperm DNA fragmentation were selected.As discussed before, we aimed to compare sperm parameters and sperm functional tests between men with a high and low degree of sperm DNA damage.These results are presented in Table 1.Statistical analysis revealed no significant differences (P > 0.05) in the mean of semen volume, sperm concentration, sperm count, abnormal morphology, and sperm intracytoplasmic ROS, between the two groups.The results indicated a significantly lower percentage of sperm motility in individuals with high sperm DNA damage, as compared to individuals with low sperm DNA damage (32% reduction, P < 0.001).Also, the mean of immature sperm chromatin packaging (assessed using aniline blue staining) was escalated by nearly twofold in individuals with high sperm DNA damage, when compared to individuals with low sperm DNA damage (80.57vs 41.59, P < 0.001).In addition, the mean percentage of sperm lipid peroxidation was significantly higher in individuals with high sperm DNA damage, as compared to the other group (21.89 vs 14.57 or 50% increase, P = 0.018).

Correlation of Omega-6 to Omega-3 fatty Acid Ratio with Sperm Parameters and Sperm Functional Tests
In the present study, we analyzed the relationship between all the types of fatty acids measured in sperm and seminal plasma with sperm parameters and its functional tests.
Considering that the main focus of this study was on the omega-3 and omega-6 fatty acids, therefore, the correlations of omega-6 to omega-3 fatty acids ratio with sperm parameters and sperm functional tests, were presented in Figs. 1  and 2, while the remaining correlations were presented in the supplementary data (Supplementary table 1 and 2).
As demonstrated in Fig. 1, the mean percentage of sperm motility showed a significant negative correlation with the omega-6 to omega-3 fatty acids ratio for both sperm and seminal plasma (R 2 = 0.36 and 0.31, respectively, P < 0.05).Statistical analysis of the remaining parameters including, sperm lipid peroxidation, sperm DNA damage assessed by TUNEL, sperm residual histones, and DNA fragmentation index assessed by SCSA, showed significant positive correlations with omega-6 to omega-3 fatty acids ratio for both sperm (R 2 = 0.13, 0.62, and 0.86, respectively, P < 0.01) and seminal plasma (R 2 = 0.09, 0.6, and 0.83, respectively, P < 0.05) (Fig. 2).
Fig. 1 The correlation between sperm and seminal plasma omega-6 to omega-3 fatty acids ratio with sperm motility

Discussion
Sperm DNA fragmentation and its relevance to fertility, especially in assisted reproduction techniques is among the most controversial issue in the field of andrology [35].The impact of DNA fragmentation on early embryonic development and the ability of the embryo to reach the blastocyst stage is becoming perceptible as the body of literature addressing this issue continues to expand in recent years.Despite numerous studies focusing on the molecular etiology of sperm DNA fragmentation, less attention has been paid to the profile of fatty acids which indirectly reflects our eating habits.It's worth mentioning that PUFAs are precursors for eicosanoids including prostaglandins and leukotrienes, which express hormone-like activities, contributing to human reproduction [36].Therefore, this study for the first time aimed to evaluate the fatty acid profile of spermatozoa and seminal plasma of men categorized by the degree of sperm DNA damage.Accordingly, individuals were selected based on a low and high degree of sperm DNA fragmentation using TUNEL and SCSA assays.
It is interesting to note that in our study, the total saturated, mono-saturated, and omega-6 fatty acids were significantly higher in both sperm and seminal plasma of individuals with high sperm DNA fragmentation.In contrast, PUFAs and omega-3 fatty acids were significantly lower in these individuals, as compared to individuals with low DNA fragmentation.Similar results were observed when individuals were compared in each lipid category (see Table 2).Expectedly, the ratio of omega-6 to omega-3 fatty acids was higher in the group with high sperm DNA fermentations.A quick look through the correlation analysis of the omega-6 to omega-3 fatty acids ratio with different sperm parameters (Fig. 1), reveals that the individuals with a low degree of sperm DNA fragmentation present a homogenous sample while those with high sperm DNA fragmentation show a wider spectrum; this indicates that the two populations are completely distinct from each other.Thus, it can be inferred that the sperm and seminal plasma lipid composition could prone sperm to DNA fragmentation which further indirectly affects sperm membrane integrity and chromatin packaging (Fig. 2).
Our results are in line with the study carried out by Martínez-Soto et al. [4].These authors showed that subjects receiving three DHA capsules per day (each capsule containing 500 mg of oil), had a significantly lower degree of sperm DNA damage, and significantly improved sperm progressive motility and anti-oxidant status, as compared to the placebo group.They did not observe significant differences in sperm concentration and morphology between the two groups [4] which is consistent with our findings (Table 1).The correlation between PUFAs and DNA fragmentation is may be related to the positive relationship previously reported between PUFA, DHA, and eicosapentaenoic acid (EPA) content of serum, sperm, and seminal plasma with serum superoxide dismutase and catalase activity [2,37].This may indicate that individuals with higher seminal PUFA content might have promoted anti-oxidant capacity or vice versa.
Another clinical trial by Safarinejad showed that DHA and EPA supplementation for 32 weeks significantly improves semen parameters, anti-oxidant capacity, superoxide dismutase, and catalase activity of seminal plasma in oligoasthenoteratospermia men, as compared to the placebo group [38].Their results are also in line with our findings that show the difference in omega-3 fatty acids value significantly affects sperm quality.Contrary to our study and aforementioned studies, Knapp showed that the nutritional supplementation of fish oil for 4 weeks altered the fatty acid composition of the spermatozoa and seminal plasma without any improvements in spermatozoa motility [39].It is worth mentioning that the inconsistent reports could be due to the difference in duration and amount of fatty acid supplementation, lifestyle, and the reproductive status of participants.These types of inconsistency results simply reflect the complexity of the addressed topic which is affected by multiple factors.In the present study, we found fewer significant correlations relative to other studies (sperm concentration and morphology), which might be due to our imitated sample size and semen volume, since we measured the entire fatty acid profile of sperm and seminal plasma separately, along with various functional sperm parameters.
The β-oxidation of fatty acids serves as one of the sources of energy for sperm motility.In this process, L-carnitine plays a central role by transporting long-chain fatty acids across the mitochondrial membranes for beta-oxidation.In a 2022 study, Iliceto et al. reported that etomoxir, an inhibitor of fatty acid oxidation, could significantly reduce sperm motility, both in vivo and in vitro.The authors added that supplementation with L-carnitine increased sperm betaoxidation and consequently improved sperm motility.Additionally, they demonstrated positive correlations between seminal plasma L-carnitine levels and total PUFAs, total omega-3 fatty acids, palmitic acid, and DHA levels; and on the other hand, negative correlations with total omega-6 PUFAs and lignoceric acid [40].
In human, during sperm maturation in the epididymis, the remodeling of sperm membrane phospholipids occurs through increasing the ratio of PUFAs level, particularly DHA, to saturated fatty acids.The significance of these events is emphasized by the fact that when group III secreted phospholipase A2 (Pla2g3) loses its functionality Fig. 2 The correlation between the ratio of omega-6 to omega-3 fatty acids in both sperm and seminal plasma, and various factors including sperm residual histones, sperm lipid peroxidation, sperm DNA damage (TUNEL assay), and the sperm DNA fragmentation index (SCSA assay) ◂ in the epididymal epithelium of mice, it leads to a lower ratio of PUFAs to saturated fatty acids, making the animal model unable to participate in fertilization [41].Interestingly, Balercia et al. demonstrated that L-carnitine supplementation reduced sperm DNA fragmentation and increased sperm motility and concentration.However, Balercia et al. did not observe a such correlation between free seminal L-carnitine and DNA fragmentation index [42].Although the above studies did not investigate the relationship between fatty acid profile and sperm DNA fragmentation, their findings may be related to the fact that sperm DNA fragmentation can occur in the testis, while L-carnitine primarily expresses its effects at post-testicular levels.This issue needs further examination.It is of note that the significance of omega-3 fatty acids in male fertility is highlighted when sperm functional tests, oxidative status, and DNA damage are examined collectively.Our results show a strong correlation between sperm motility and both sperm and seminal omega-6 to omega-3 fatty acids ratio (Fig. 1), which is consistent with previous reports [8,43,44] that also demonstrated a strong correlation between human sperm motility and spermatozoa membrane DHA levels.Moreover, a direct relationship between DHA concentrations in spermatozoa and fertility has been demonstrated in an animal model [11].This observation is likely due to the anti-oxidant properties of omega-3 fatty acids [45] and the effect that PUFAs have on spermatozoa membrane fluidity and thereby fertilization [46].In this regard, the level of seminal PUFAs is critical for successful sperm capacitation [47].In agreement with the findings of the present study, Safarinejad and Safarinejad showed that total omega-3 fatty acids, a-linolenic acid, EPA, and DHA concentrations are positively associated with sperm concentration, motility, morphology, and anti-oxidant activity of seminal plasma [37].Similarly, Attaman confirmed that increased dietary intake of omega-3 PUFAs was positively correlated with sperm morphology [30].
It has been shown that myristic acid facilitates the conversion of α-linolenic acid to DHA and then to other PUFAs in sperm cells [48,49].Considering that PUFAs are the main constituent of the membranes of spermatogonia and spermatozoa, therefore, Khalil et al. concluded that myristic acid helped to preserve the integrity of sperm membrane from oxidative damage in testicular tissue of diabetes-induced rats [48].However, in the present study, we did not observe any significant difference in myristic acid levels between individuals with a low and high degree of DNA fragmentation.This inconsistency with the findings of Khalil et al. is likely due to differences in experimental design between the two studies.Another possibility is that in the study by Khalil et al., myristic acid inhibited the effect of hyperglycemia on the AGE/RAGE pathway, which stimulates the production of cytokines and growth factors, resulting in damage to spermatozoa [48].This mechanism may differ from the etiology of DNA fragmentation in our study.

Limitation
One of the limitations of this study is the lack of consideration of the effect of lifestyle factors on sperm parameters and fatty acid profile.Since the main objective of the study was to evaluate the relationship between plasma and sperm fatty acid profiles with sperm parameters and DNA status, we underscore the significance of future studies that assess the impact of lifestyle factors on fatty acid profiles in both sperm and seminal plasma.

Conclusion
Men with high levels of sperm DNA fragmentation often exhibit a low percentage of sperm motility, increased oxidative stress, abnormal sperm chromatin packaging, and an imbalance in the ratio of omega-6/omega-3 fatty acids.Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better quality sperm function compared to men with a lower omega-3 fatty acid.

Table 2
The comparison of fatty acid composition between sperm and seminal plasma samples from men with high and low DNA damage1