Infertility is a significant global health problem, with male factors contributing to approximately 50% of all cases. ¹ Despite that several causes, including hormonal abnormalities, genetic diseases, infection, and exposure to environmental pollutants have been identified, many of cases are still idiopathic, even if the patient has normal semen parameters. ² Classic semen analysis assesses concentration, motility, and morphology, but these parameters do not always reflect the functional and molecular competence of spermatozoa. ³

Among the ART procedures, ICSI has been one of the most commonly used techniques for the treatment of male factor infertility especially when routine semen characteristics are severely deranged. Although ICSI bypass many physiological barriers to fertilization, it cannot correct pre-existing DNA damage due to oxidative stress (OS). It is shown that sperm with highly fragmented DNA could have normal morphology and motility. ^{4, 5} In this regard, conventional selection criteria does not exclude the injection of a spermatozoon with high DNA damage. This limitation underscores the need for molecular-level indicators to enhance ICSI outcome prediction.

OS is among the most studied factors associated with male infertility. ^{6, 7} Excess production of reactive oxygen species (ROS) or inefficient scavenging may lead to lipid, protein, and DNA damage, affecting sperm viability and function. ^{8, 9} In high concentrations, ROS negatively impact sperm capacitation and fertilization by causing mitochondrial injury, membrane instability, and DNA damage of the male gamete. ¹⁰

Seminal plasma creates a protective and antioxidant milieu which is crucial to protect sperm function. ^{11, 12} Seminal total antioxidant capacity (TAC) reflects the overall activity of all antioxidants within seminal fluid and is used as an OS status marker. TAC has found to be substantially lower in infertile men, ¹³ particularly in oligoasthenoteratozoospermic (OAT) men, as compared to fertile individuals. ¹⁴

Seminal plasma cell-free DNA (cfDNA) has recently been proposed as a promising biomarker. This DNA consists of extracellular DNA fragments that are present in seminal plasma through several means, apoptosis, necrosis, active cellular excretion, and neutrophil extracellular trap formation (NETosis). ^{15, 16} The existence of cfDNA in semen was first demonstrated in 2004 by Chou and coworkers, who described it as being of two major types: low molecular weight (1 kb) DNA and high molecular weight (~12 kb) DNA. ¹⁷ Later studies showed that cfDNA from seminal plasma is of a wide range size (180 bps to 15 kb) indicating various types of cellular origin and release methods. ^{18, 19} Seminal cfDNA has been associated with reduced sperm quality and higher DNA fragmentation. ^{20, 21} As a non-cellular biomarker, cfDNA could offer a non-invasive means to evaluate sperm genome integrity and complements traditional semen parameters in fertility assessment.

While OS markers like TAC and cfDNA have been correlated with sperm dysfunction, their influence on ICSI outcomes remain inadequately explored. Although pregnancy rates represent the primary measure of ART success in several studies, the utility of miscarriage rates may be more revealing. As ICSI bypasses natural selection processes that occurs during normal fertilization, even DNA-damaged sperm may fertilise an oocyte. ^{22– 24} Hence, assessing miscarriage rates offers an appropriate estimation of post-fertilization embryo viability and the effects of oxidative DNA damage.

This study aimed to assess the levels of cfDNA and TAC in the seminal plasma of males undergoing ICSI. It is also sought to evaluate the correlation between these markers and ICSI outcomes including embryo grading, fertilization rate, pregnancy rate, and miscarriage rate, and to investigate their relationship with conventional semen parameters such as sperm concentration, motility, and morphology.

The current study included 65 couples undergoing ICSI. Among them, 45 couples diagnosed with male factor infertility were categorized into the sperm disorder group, while the remaining 20 couples, who demonstrated normal semen parameters, comprised the normospermia group.

There was a statistically significant difference regarding the age-distribution between groups as demonstrated in Table 1 ( p = 0.034). A higher proportion of men aged ≥40 years was observed in the sperm disorder group (35.6%) compared to the normospermia group (5.0%), while the 25–29 age group was more prevalent among normospermic males (50.0% vs. 22.2%). The mean age of the sperm disorder group was also significantly higher (36.1 ± 6.4 years) than that of the normospermia group (30.6 ± 4.7 years, p = 0.001). Although the average BMI in the sperm disorder group was slightly higher, the difference was not significant. Additionally, 55.6% of the sperm disorder group was smoker, while no one in the normospermia group was smoker.

The duration of sterility was assessed in the sperm disorder group, showing an average of 6.93 ± 3.04 years, with a range between 3 and 15 years. More than half (51.1%) had experienced sterility 5–9 years, followed by 10–15 years (26.7%) and lastly 1–4 years (22.2%).

Significant variations in seminal biomarkers were evident between the two groups. As shown in Table 2, the mean cfDNA concentration was substantially elevated in the sperm disorder group (3.482 ± 0.936 μg/ml) compared to the normospermia group (0.631 ± 0.454 μg/ml, p = 0.0001). In contrast, TAC was significantly lower in the sperm disorder group (1260.42 ± 251.29 μM/ml) than in the normospermia group (1847.80 ± 70.71 μM/ml, p = 0.0001). These findings strongly suggest that increased OS and cfDNA are closely linked to impaired semen quality.

Further analysis ( Table 3, Figures 1 & 2) revealed notable differences in cfDNA and TAC levels across various types of sperm abnormalities. Men with OAT exhibited the highest cfDNA concentration (4.476 ± 0.552 μg/ml), followed by those with asthenospermia (3.850 ± 0.922 μg/ml), teratospermia (3.120 ± 0.529 μg/ml), and oligospermia (2.781 ± 0.735 μg/ml). These differences were highly significant ( p = 0.0001, ANOVA).

Conversely, TAC levels showed a decreasing trend with increasing severity of sperm abnormalities. The highest TAC was observed in the oligospermia group (1490.93 ± 194.08 μM/ml), followed by teratospermia (1309.58 ± 127.73 μM/ml), asthenospermia (1167.89 ± 172.62 μM/ml), and the lowest in the OAT subgroup (962.00 ± 120.43 μM/ml). These differences were also statistically significant ( p = 0.0001, ANOVA), supporting an inverse relationship between OS and semen quality.

Building on these biomarker disparities, Table 4 explores how cfDNA correlates with ICSI outcomes. A clear and consistent trend emerged: higher cfDNA levels were associated with lower-quality embryos. In the sperm disorder group, grade I embryos were linked to the lowest cfDNA concentration (2.195 ± 0.66 μg/ml), while grade III embryos had the highest (4.023 ± 0.85 μg/ml, p = 0.0001). This pattern was also observed in the normospermia group but at significantly lower cfDNA levels.

The impact of cfDNA on fertilization success followed a similar pattern. In the normospermia group, men with successful fertilization had markedly lower cfDNA levels (0.474 ± 0.34 μg/ml) compared to those with failed fertilization (1.257 ± 0.27 μg/ml, p = 0.0001). Although this trend was also seen in the sperm disorder group, it did not reach statistical significance.

Pregnancy outcomes further substantiated cfDNA’s predictive value. In the sperm disorder group, men whose partners achieved pregnancy had lower cfDNA levels (2.90 ± 1.15 μg/ml) compared to those who did not conceive (3.75 ± 0.80 μg/ml, p = 0.017). Similarly, in the normospermia group, successful pregnancies were associated with significantly lower cfDNA levels (0.372 ± 0.24 μg/ml) than unsuccessful ones (0.917 ± 0.39 μg/ml, p = 0.007). Miscarriages were also linked to elevated cfDNA concentrations in both groups. In the sperm disorder group, the mean cfDNA level was (3.230 ± 1.08 μg/ml) in miscarriage cases, compared to (1.768 ± 0.46 μg/ml) in pregnancies that progressed ( p = 0.048). In the normospermia group, miscarriage cases had a mean cfDNA level of (0.675 ± 0.03 μg/ml), while ongoing pregnancies showed lower levels (0.317 ± 0.22 μg/ml, p = 0.049). These findings suggest that excessive sperm DNA fragmentation may compromise embryo viability even after implantation.

To complement cfDNA findings, Table 5 evaluates the role of TAC in ICSI success. Consistently, higher TAC levels correlated with favorable outcomes. For instance, among sperm disorder cases, grade I embryos were associated with the highest TAC (1549.42 ± 191.06 μM/ml), significantly surpassing those linked to grade III embryos (1137.15 ± 203.98 μM/ml, p = 0.017). A similar relationship was evident in the normospermia group, reinforcing the protective role of antioxidants in embryogenesis.

Regarding fertilization, TAC levels were slightly higher in successful cases within both groups. In the sperm disorder group, fertilized cases had a mean TAC of (1264.56 ± 249.49 μM/ml), compared to (1247.64 ± 268.71 μM/ml) in non-fertilized cases, though this difference was not statistically significant ( p = 0.849). In the normospermia group, fertilized cases had a mean TAC of (1849.81 ± 73.21 μM/ml), while non-fertilized cases showed a slightly lower value of (1839.75 ± 69.014 μM/ml); again, the difference was not statistically significant ( p = 0.807).

When examining pregnancy outcomes, TAC levels were again higher in successful pregnancies. In the sperm disorder group, pregnancies were associated with a mean TAC of (1370.23 ± 255.06 μM/ml), significantly higher than the (1199.14 ± 227.86 μM/ml) observed in non-pregnant cases (p = 0.050). On the other hand, the normospermia group showed no significant difference in TAC levels between successful pregnancies (1843.15 ± 71.29 μM/ml) and unsuccessful cases (1878.66 ± 90.33 μM/ml; p = 0.468).

Furthermore, miscarriage analysis revealed that in the sperm disorder group, pregnancies that progressed successfully had significantly higher TAC levels (1628.66 ± 224.02 μM/ml) compared to miscarried cases (1292.70 ± 215.98 μM/ml, p = 0.039). In the normospermia group, a similar significant difference was noted, with ongoing pregnancies having a mean TAC of (1859.72 ± 64.289 μM/ml), compared to (1752.00 ± 5.657 μM/ml) in miscarriage cases ( p = 0.043).

Finally, Table 6 presents the correlations between cfDNA, TAC, and conventional semen parameters. In the sperm disorder group, cfDNA levels showed significant negative correlations with progressive motility (grades A and B; p < 0.01) and a significant positive correlation with immotile sperm (grade D; p = 0.0001). Although a negative association was observed between cfDNA and sperm morphology, it did not reach statistical significance ( p = 0.088). No significant correlations were found between cfDNA and sperm concentration, age, or BMI.

Conversely, TAC levels demonstrated significant positive correlations with progressive motility (grades A and B; p < 0.001) and morphology ( p = 0.003), along with a significant negative correlation with immotile sperm (grade D; p = 0.0001). However, TAC levels showed no significant correlation with sperm concentration, age, or BMI.

In the normospermia group, none of the correlations between cfDNA or TAC and semen parameters, age, or BMI reached statistical significance, likely due to the relatively uniform semen quality and demographic characteristics within that cohort.

The current study included 65 couples who were candidates for ICSI. Of these, 45 couples, who had male factor infertility, were assigned to the sperm disorder group, while 20 couples constituted the normospermia group, exhibiting normal semen parameters. For consistency, couples in which the female partner had normal fertility evaluation were selected to avoid potential confounder of female infertility. Female partners who had any documented reproductive abnormalities, such as endometriosis, diminished ovarian reserve, and other gynecological conditions, were not included in the study. This model was used to concentrate on the impact of male seminal factors, specifically levels of cfDNA and TAC on ICSI outcomes. The difference in fertilization rates and pregnancy/miscarriage outcomes were compared between two male groups to explore the effects of sperm quality and OS indicators on the success of assisted reproduction.

Demographic analysis of the study population ( Table 1) revealed a significant difference between the groups in the mean male age. The mean age in the sperm disorder group was significantly higher than that of the normospermia group. However, it is important to note that the average duration of sterility among males in the sperm disorder group was 6.9 ± 3.04 years. This indicates that the sperm abnormalities could present at a younger age and were not a direct consequence of aging. Therefore, while advancing age is an important factor influences semen quality, ^{25, 26} the early onset of infertility in those males suggest that intrinsic seminal defects played a major role in their reproductive dysfunction. Importantly, the effect of paternal age on ICSI outcomes remains controversial. Some studies have stated that the paternal age has a negative correlation with ICSI success rate, ^{25, 27, 28} while others have found that the advanced paternal age has no effect on the outcomes of ICSI cycles. ^{29, 30}

The analysis of BMI revealed no significant difference between the two groups. The mean BMI for sperm disorder group was (26.80 ± 3.51 kg/m ²) compared to (25.93 ± 2.98 kg/m ²) in the normospermia group ( p = 0.342). The comparable BMI values suggest no appreciable contribution of the weight status to the differences observed in sperm parameters, semen biomarkers, and ICSI outcomes.

Regarding smoking status in the study population, all participants were non-smokers in the normospermia group, whereas a large proportion (55.6%) in the sperm disorder group reported smoking. Studies have demonstrated that smoking compromises sperm quality by increasing OS, increasing DNA damage, and inducing cell apoptosis. ^{31– 33} Therefore, smoking could contribute partially to the observed differences in seminal oxidative balance and ICSI outcomes.

Our study demonstrated that men with sperm abnormalities exhibited significantly elevated levels of seminal cfDNA compared to normospermic individuals (3.482 ± 0.936 μg/ml vs. 0.631 ± 0.454 μg/ml; p = 0.0001), as shown in Table 2. This finding is in agreement with several studies that reported a significant relationship between increased levels of seminal cfDNA and subnormal sperm parameters in infertile men. According to Javidmehr et al. (2024), ³⁴ higher cfDNA levels are consistently found in men with sperm abnormalities such as oligozoospermia, teratozoospermia, and azoospermia, compared to normozoospermic controls. These findings were supported by the work of Mbaye et al. (2021) ³⁵ who reported that significantly elevated cfDNA levels in men with poor sperm count, morphology, and high DNA fragmentation index (DFI), suggesting that cfDNA reflects underlying sperm chromatin instability and apoptosis. Similarly, Di Pizio et al. (2020) ²¹ found that cfDNA concentrations were markedly elevated in men with sperm disorders compared to normospermic men (2.09 μg/mL versus 1.18 μg/mL), and positively correlated with the severity of sperm defects. The work by Costa et al. (2017) ³⁶ also highlighted that cfDNA levels (measured by PicoGreen fluorochrome) correlated inversely with standard semen quality metrics such as motility and morphology.

It is noteworthy to mention that cfDNA could be released into the seminal plasma by different biological processes, mainly apoptosis, necrosis, and a particular process named NETosis. ¹⁶ These cellular events often occur in response to abnormal spermatogenesis or impaired maturation processes, particularly in men with oligozoospermia, teratozoospermia, or azoospermia. Di Pizio et al. (2020) ²¹ observed that men with teratozoospermia exhibited elevated cfDNA levels than men with normal sperm parameters. They proposed that spermatogenic impairment may activate cellular checkpoints that lead to apoptotic DNA fragmentation and subsequent release of cfDNA. The released cfDNA itself could have a detrimental effect on sperm parameters by triggering sperm DNA fragmentation. Aitken et al. (2020) ³⁷ proposed that extracellular free DNA can induce DNA fragmentation in sperm through nuclease activation, thereby contributing to a feedback loop of genetic damage and infertility.

The role of OS as a contributor to elevated levels of cfDNA was demonstrated experimentally by Costa et al. (2017). ³⁶ They demonstrated that exposing semen samples to paraquat, a ROS inducer, led to a significant increase in cfDNA levels, coupled with reduced sperm viability, motility, and morphology. These results suggest that cfDNA not only originates from sperm degeneration but also serves as a sensitive indicator of oxidative damage in the male reproductive system.

Spermatozoa are uniquely vulnerable to oxidative damage due to high polyunsaturated fatty acid (PUFA) content in their membranes, scarce cytoplasmic volume, limited DNA repair capacity, and, in defective sperm, improper chromatin packaging that exposes DNA to oxidative damage. ^{38, 39} According to Oliva et al. (2006), ⁴⁰ infertile men exhibited elevated histone: protamine ratio, which results in insufficient chromatin compaction and increases vulnerability to oxidative damage. A finding by Sakkas and Alvares ⁴¹ emphasizes that oxidative injury to spermatozoa results in increased DNA strand breaks, which may subsequently impair fertilization, embryo development, and pregnancy.

Our study demonstrated a significantly lower TAC in men with sperm abnormalities compared to normospermic men (1260.42 ± 251.29 μM/ml vs. 1847.80 ± 70.71 μM/ml; p = 0.0001), as shown in Table 2. This decline reflects a compromised antioxidant defense system in the sperm disorder group, underscoring the role of OS in male infertility. As a cumulative parameter of both enzymatic and non-enzymatic antioxidants in seminal plasma, TAC reflects the body’s ability to neutralize ROS, which are known to cause lipid peroxidation, DNA fragmentation, and reduced sperm viability. This finding supports Tremellen’s (2008) ⁴² OS model of male infertility, which postulates that when ROS generation exceeds antioxidant neutralization, oxidative damage undermines reproductive potential.

This mechanism is strongly supported by findings from Pahune et al. (2013), ⁴³ Giulini et al. (2009), ⁴⁴ and Demir and Özdem (2022), ⁴⁵ who demonstrated that infertile men, especially those with combined sperm abnormalities exhibited significantly lower TAC levels than normospermic individuals, aligning well with our observations.

Additional corroboration comes from Saleh et al. (2018), ¹³ who showed that infertile men had significantly lower TAC compared to healthy men. In addition, they reported that infertile men have a significant high level of caspase-3 activity, an indicator of apoptosis, implying that oxidative damage may trigger cell death pathways detrimental to spermatogenesis. Further, Alahmar et al. (2021) ⁴⁶ emphasized that men with idiopathic oligoasthenozoospermia show particularly diminished TAC levels and high DNA fragmentation, underscoring the central role of OS in their pathophysiology.

The concurrent elevation of cfDNA and reduction of TAC in our sperm disorder group reinforces this mechanistic link, suggesting that OS not only contributes to defective spermatogenesis but may also mediate DNA instability and impaired fertilization potential. The reduced TAC likely plays a pivotal role in the poor semen quality and ICSI outcomes seen in this cohort.

A stratified subgroup analysis was conducted to investigate the differences in cfDNA and TAC across groups of sperm abnormalities, as shown in Table 3, Figures 1 & 2. Oligospermia was associated with a cfDNA concentration of (2.78 ± 0.73 μg/ml) and the highest TAC among the disorder groups at (1490.93 ± 194.08 μM/ml), indicating relatively limited oxidative disruption. These results are in line with findings by Mbaye et al. (2021), ³⁵ who reported elevated cfDNA levels in oligospermic men (1.26 ± 0.22 μg/ml) compared to normospermic controls (0.09 ± 0.01 μg/ml). In terms of antioxidant defense, Eroglu et al. (2014) ⁴⁷ similarly observed significantly reduced TAC in oligospermic patients compared to normospermic individuals, suggesting that even isolated sperm count deficiencies are linked to early OS and DNA damage.

In teratospermia, cfDNA levels reached (3.12 ± 0.52 μg/ml), while TAC dropped to (1309.58 ± 127.72 μM/ml). These findings reflect increased oxidative damage and are supported by Di Pizio et al. (2020), ²¹ who documented significantly higher cfDNA in teratozoospermic men (1.80 ± 0.33 μg/ml) than in normospermic individuals (1.29 ± 0.26 μg/ml). Regarding antioxidant status, Demir and Özdem (2022) ⁴⁵ also reported lower TAC levels in men with teratospermia (median 1.57 mmol/L) compared to fertile controls (1.74 mmol/L), reinforcing the association between morphological sperm defects and oxidative imbalance.

In asthenospermia, cfDNA increased further to (3.85 ± 0.92 μg/ml), and TAC declined to (1167.88 ± 172.62 μM/ml), reflecting compromised sperm motility and redox status. Mbaye et al. (2021), ³⁵ observed that the level of cfDNA in males with asthenozoospermia is significantly higher than normospermic males. Additionally, Alahmar et al. (2021) ⁴⁶ found significantly elevated DFI and reduced TAC levels in men with poor motility, suggesting a shared oxidative mechanism. It was found that ROS-induced mitochondrial DNA damage reduces ATP production and energy supply, thereby impairing sperm motility. ⁴⁸ This is further supported by Khosrowbeygi and Zarghami (2008), ⁴⁹ who reported a positive correlation between TAC and sperm motility, implying that reduced antioxidant defenses underlie motility impairments.

The most severe profile was observed in men with OAT, who had the highest seminal cfDNA levels at (4.47 ± 0.55 μg/ml) and the lowest TAC values at (962.00 ± 120.43 μM/ml). These values reflect a compounded oxidative burden resulting from the simultaneous presence of three sperm abnormalities. It has been reported that abnormal sperms tend to generate ROS and further compromising the seminal antioxidant defense. ^{9, 48} Javdimehr et al. (2025) ⁵⁰ reported that men with OAT had significantly higher seminal cfDNA levels compared to those with oligoasthenospermia, oligoteratozoospermia, nonobstructive azoospermia, and normozoospermia. Although Di Pizio et al. (2021) ²¹ did not specifically analyze OAT, they did report that azoospermic patients, often considered even more severe, had cfDNA levels as high as (3.65 ± 0.88 μg/ml), reinforcing the trend of escalating cfDNA with increasing pathology. For TAC, Demir and Özdem (2022) ⁴⁵ showed that OAT patients had the lowest median TAC (1.19 mmol/L), and Giulini et al. (2009) ⁴⁴ reported even more dramatic reductions in moderate (0.83 ± 0.36 mmol/L) and severe (0.70 ± 0.43 mmol/L) OAT cases.

Although direct DNA fragmentation testing was not performed in this study, the elevated cfDNA levels in the sperm disorder group provide a strong indirect marker. cfDNA is released from apoptotic and DNA-damaged sperm and has been correlated with DFI in infertile men. This relationship is confirmed by several studies which demonstrated that patients with elevated DNA fragmentation had significantly higher levels of seminal cfDNA, supporting the validity of cfDNA as a surrogate marker for sperm genomic instability. ^{20, 21, 37} Furthermore, the sperm disorder group exhibited significantly reduced TAC, reinforcing the presence of OS as a contributing factor. As Agarwal et al. (2020) ⁵¹ and Giulini et al. (2009) ⁴⁴ have shown, oxidative damage is a major mechanism behind chromatin instability, with reduced TAC directly linked to impaired fertilization and embryo development.

The relationship between sperm oxidative biomarkers and ICSI outcomes is clearly illustrated in Tables 4 & 5. Our study reveals that fertilization rates were statistically comparable between the normospermia group (80.0%) and the sperm disorder group (75.6%) ( p = 0.695). The lack of significant difference in fertilization rates despite differing sperm quality profiles can be explained by the nature of ICSI, where natural selection processes are bypassed. Sperms are selected based on motility and morphology under the microscope, which does not reveal their underlying genomic integrity. As noted by Aitken and De Iuliis (2007), ⁵² ICSI enables the fertilization of oocytes using sperm that may be genomically compromised. Bungum et al. (2007) ²² added that even sperm with DNA damage can successfully form pronuclei and initiate early embryonic development in ART settings, though their developmental potential may be impaired. This view is supported by Sun et al. (2018) ²³ and Al Omrani et al. (2018), ²⁴ who reported that high sperm DNA fragmentation had no statistically significant effect on fertilization rates in ICSI procedures, reinforcing the idea that fertilization may proceed despite compromised DNA integrity. Similarly, Henkel et al. (2003), ⁵³ Li et al. (2024), ⁵⁴ and Sivanarayana et al. (2014) ⁵⁵ reported that high DNA fragmentation does not significantly impair fertilization rates in ICSI, further supporting the notion that fertilization may be an inadequate endpoint for assessing the true reproductive consequences of genomic instability, which may manifest later during embryo development or implantation.

However, other studies present contrasting evidence: Lopes et al. (1998) ⁵⁶ and Huang et al. (2005) ⁵⁷ found that increased DNA fragmentation was negatively associated with fertilization success, suggesting that extensive genomic damage may still hinder ICSI outcomes at this early stage. Virro et al. (2004) ⁵⁸ observed that DNA fragmentation levels above 30% were associated with significantly reduced fertilization rates in IVF, emphasizing that the fertilization outcome may be more sensitive to DNA quality in procedures where natural selection remains partially intact. Additionally, a study by Ribas-Maynou et al. (2022) ⁵⁹ reported that sperm DNA fragmentation reduces fertilization rates and delays embryo development in ICSI cycles.

While ICSI can overcome mechanical barriers to fertilization, it does not eliminate the impact of sperm DNA damage on embryo development and pregnancy. It has been demonstrated that DNA-fragmented sperm may appear morphologically normal and motile, increasing the chance of being selected during ICSI and potentially contributing to compromised embryo development and early pregnancy loss. ^{5, 60}

According to our study, embryo quality was inversely related to cfDNA levels Table 4. Grade I embryos were associated with the lowest cfDNA concentration (2.19 ± 0.662 μg/ml), while grade III embryos were linked to the highest levels (4.02 ± 0.858 μg/ml; p = 0.0001). This finding is consistent with Ribas-Maynou et al. (2022), ⁵⁹ who observed that sperm DNA damage negatively impacts blastocyst formation and embryo development. Similarly, Simon et al. (2011) ⁶¹ reported that elevated sperm DNA fragmentation is associated with the generation of poor-quality embryos, thereby reducing the likelihood of achieving successful implantation. Seli et al. (2004) ⁶² found that high levels of sperm DNA fragmentation adversely affected blastocyst development in IVF cycles, highlighting the relevance of sperm genomic integrity during early embryogenesis. In addition, Virro et al. (2004) ⁵⁸ demonstrated that high sperm DNA damage had reduced blastocyst formation rates in IVF/ICSI cycles. Borini et al. (2006) ⁶³ further emphasized that sperm DNA damage compromises embryo quality even post-implantation, potentially affecting later developmental stages in ICSI-derived pregnancies. Moreover, Henkel et al. (2003) ⁵³ explained that spermatozoa with high DNA fragmentation may appear functionally competent during fertilization but later impair embryonic progression once the paternal genome becomes transcriptionally active, offering a mechanistic rationale for the observed reduction in embryo quality.

Seminal cfDNA levels also demonstrated strong predictive value for pregnancy success. In the sperm disorder group, men whose partners achieved pregnancy had significantly lower cfDNA levels than those who did not (2.90 ± 1.153 vs. 3.75 ± 0.801 μg/ml; p = 0.017). A similar trend was observed in the normospermia group (0.372 ± 0.242 vs. 0.917 ± 0.393 μg/ml; p = 0.007), underscoring the reliability of cfDNA as a marker of sperm genomic integrity. Our findings align with previous research demonstrating that elevated sperm DNA fragmentation is negatively associated with pregnancy rates in ICSI cycles. ^{64– 69}

Importantly, unlike many previous studies that conclude ICSI success at the point of achieving clinical pregnancy, our investigation extended the follow-up period to include miscarriage outcomes. We believe that clinical pregnancy alone may be an insufficient endpoint, as it does not capture the full trajectory of reproductive success. By monitoring participants through early gestation, we were able to evaluate the risk of pregnancy loss, a more definitive and biologically meaningful outcome of ICSI. This approach allowed us to link seminal oxidative markers, such as cfDNA and TAC, not only to fertilization and implantation but also to the ability to sustain a viable pregnancy. Consequently, miscarriage rate in our view represents a more comprehensive and clinically relevant indicator of ICSI outcome.

In our study, miscarriage outcomes showed a strong link to cfDNA levels. Table 4 indicates that ongoing pregnancies were associated with significantly lower cfDNA levels (1.76 ± 0.466 μg/ml) than pregnancies that ended in loss (3.23 ± 1.085 μg/ml; p = 0.048). These findings mirror those reported by Zini et al. (2008) ⁷⁰ and Virro et al. (2004), ⁵⁸ who showed that sperm DNA fragmentation is a key predictor of miscarriage following ART procedures. Further support comes from Benchaib et al. (2003), ⁶⁸ who found that DFI greater than 15% were significantly associated with elevated miscarriage risk in ICSI patients. Similarly, Boe-Hansen et al. (2006), ⁷¹ demonstrated that while clinical pregnancy can still be achieved, the likelihood of carrying to term may be reduced when DFI approaches or exceeds 27%. Additionally, Lin et al. (2008), ⁷² highlighted that DNA fragmentation exerts its most pronounced effect during post-implantation stages, significantly increasing miscarriage rates while showing limited influence on initial pregnancy establishment. As we mentioned earlier, in ICSI procedure, the sperm selection is based on morphology and motility, which do not reflect its genomic integrity. In a meta-analysis study, Zhao et al. (2014), ⁷³ reported a strong association between elevated sperm DNA fragmentation and increased miscarriage rates in ICSI, but not IVF, highlighting the consequences of bypassing natural sperm selection.

The mechanism by which DNA damage contributes to pregnancy failure may be delayed in its manifestation. It has been reported that the embryonic genome remains transcriptionally silent until after the second cleavage division, suggesting that abnormalities in the paternal genome may not influence fertilization or early embryonic development but become apparent at later stages. ⁷⁴ As such, sperm with substantial DNA damage may successfully fertilize oocytes and form early-stage embryos in vitro, yet fail to support proper development in utero, ultimately leading to miscarriage.

TAC levels were also associated with key ICSI outcomes, as shown in Table 5. Higher TAC was significantly associated with better embryo quality, as men whose partners produced grade I embryos had the highest mean TAC (1549.42 ± 191.067 μM/ml), while grade III embryos were linked to the lowest TAC (1137.15 ± 203.988 μM/ml; p = 0.017). These results are in line with Greco et al. (2005), ⁷⁵ who found that antioxidant treatment reduced sperm DNA fragmentation and improved embryo morphology in ICSI cycles. Scaruffi et al. (2021) ⁷⁶ similarly reported enhanced blastocyst development and pregnancy rates in antioxidant-treated men undergoing ART.

While TAC did not significantly influence fertilization rates, it was predictive of pregnancy. In the sperm disorder group, TAC was higher in those who achieved pregnancy (1370.23 ± 255.063 μM/ml) compared to non-pregnant individuals (1199.14 ± 227.860 μM/ml; p = 0.050). Miscarriage data showed even more pronounced differences: ongoing pregnancies were associated with significantly higher TAC (1628.66 ± 224.020 μM/ml) than those that ended in loss (1292.70 ± 215.984 μM/ml; p = 0.039). This pattern was similarly observed in the normospermia group ( p = 0.043). These findings align with Gupta et al. (2021) ⁷⁷ and Alahmar et al. (2018), ⁷⁸ who documented the relationship between oxidative imbalance, reduced TAC, and increased risk of early pregnancy loss.

Together, these findings underscore that TAC is not only a marker of sperm quality but also a predictive indicator of ICSI success. Given that antioxidant systems in seminal plasma function to neutralize ROS and protect sperm DNA from oxidative damage, their depletion can compromise reproductive outcomes. As supported by Agarwal et al. (2020) ⁵¹ and Tremellen (2008), ⁴² correcting this imbalance through antioxidant therapies or lifestyle modifications may enhance embryo viability, reduce miscarriage, and improve ART success rates, especially in men with high OS profiles.

In the sperm disorder group, cfDNA levels demonstrated significant negative correlations with progressive motility types A and B (r = –0.491, p = 0.0001; r = –0.439, p = 0.003) and a strong positive correlation with immotile sperm (type D) (r = 0.621, p = 0.0001) ( Table 6). These results indicate that higher cfDNA, suggestive of greater DNA fragmentation, is associated with impaired sperm motility. This relationship aligns with the findings of Benchaib et al. (2003), ⁶⁸ Appasamy et al. (2007), ⁷⁹ Le et al. (2019), ⁸⁰ and Al Omrani et al. (2018), ²⁴ who documented that increased DNA fragmentation correlates negatively with sperm motility. Costa et al. (2017) ³⁶ further linked high seminal cfDNA to motility and morphology impairments.

However, cfDNA did not show statistically significant correlations with sperm concentration or morphology. This is consistent with Liu et al. (2023) ⁸¹ and Al Omrani et al. (2018), ²⁴ who found no association between DNA damage and morphology. Nevertheless, Liu et al. (2023) ⁸¹ and Al Omrani et al. (2018) ²⁴ reported negative correlations between DFI and concentration. Similarly, Le et al. (2019) ⁸⁰ did observe a negative association between DNA damage and sperm head morphology, suggesting variability across cohorts.

Regarding demographic parameters, cfDNA levels were not significantly correlated with age and BMI ( Table 6). However, contrasting evidence was reported by Shi et al. (2018), ⁸² Das et al. (2013), ⁸³ Rybar et al. (2011), ⁸⁴ and Chua et al. (2023), ⁸⁵ all of whom found a positive correlation between age and DNA fragmentation. Likewise, studies by Kort et al. (2006), ⁸⁶ La Vignera et al. (2012), ⁸⁷ and Fariello et al. (2012) ⁸⁸ reported a positive correlation between BMI and DFI.

TAC levels in the sperm disorder group showed significant positive correlations with progressive motility types A and B (r = 0.496 and 0.550, both p = 0.0001) ( Table 6), and with sperm morphology (r = 0.434, p = 0.003), while showing a strong inverse correlation with immotile sperm (type D) (r = –0.700, p = 0.0001). These findings support the antioxidant role of seminal plasma in preserving motility and structural integrity. Our results align with previous research from Khosrowbeygi et al. (2007), ⁸⁹ Koca et al. (2003), ⁹⁰ Demir & Ozdem (2022), ⁴⁵ Pahune et al. (2013), ⁴³ Gupta et al. (2020), ⁷⁷ Eroglu et al. (2014), ⁴⁷ and Giulini et al. (2009), ⁴⁴ all of whom found that TAC is positively correlated with motility and morphology.

TAC showed no significant correlations with sperm concentration, age, and BMI, which is consistent with Appasamy et al. (2007) ⁷⁹ and El-Gazzar et al. (2023). ⁹¹ Nonetheless, Gupta et al. (2020), ⁷⁷ Giulini et al. (2009), ⁴⁴ and Eroglu et al. (2014) ⁴⁷ did report a positive association between TAC and sperm concentration.

A strong negative correlation was observed between cfDNA and TAC (r = –0.785, p = 0.0001), supporting the role of OS in compromising DNA integrity. This is in agreement with Liu et al. (2023), ⁸¹ who demonstrated an inverse association between TAC and DNA fragmentation, and indirectly supported by the OS model proposed in studies like Costa et al. (2017). ³⁶

Lastly, in the normospermia group, no significant correlations were detected between cfDNA, TAC, and semen parameters ( Table 6). This may reflect the relatively stable redox environment and lower variability in seminal biomarkers among men with normal semen profiles, limiting the detectability of such associations in this group.

This study has some limitations that should be considered when interpreting the results. First, it was conducted at a single center with a relatively modest sample size, which may limit the generalizability of the findings to broader populations. Second, although seminal cfDNA was employed as a surrogate marker, direct measurement of the sperm DFI would have provided more robust validation of DNA integrity assessments. Finally, potential confounding variables such as lifestyle habits, occupational exposures, dietary patterns, and psychological stress were not controlled, all of which may impact OS levels and antioxidant status.

The present work underscores the importance of seminal OS, manifested by increased cfDNA and decreased TAC, on the pathophysiology of male infertility and ICSI outcome. Our findings reveal that sperm abnormalities are associated with significant oxidative imbalance, which in turn correlates with impaired sperm motility, morphology, and, importantly, poor ICSI outcomes such as reduced pregnancy rates and elevated miscarriage incidence. cfDNA and TAC could be valuable predictive markers for embryo quality, pregnancy success, and miscarriage risk. Taken together, these findings support the incorporation of cfDNA and TAC measurements into the practice of diagnostic fertility testing, thereby providing a more comprehensive and clinically relevant representation of male reproductive potential and facilitating a highly individualized treatment strategy in ART.