An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = ?.586, r = ?.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.     

Seminal oxidation–reduction potential levels are not influenced by the presence of leucocytospermia

Oxidative stress (OS) is characterised by an excessive amount of reactive oxygen species (ROS) which negatively affect sperm functions. In this study, the influence of leucocytes on seminal oxidation–reduction potential (ORP) and sperm DNA fragmentation (SDF) was investigated in 1,068 men. Seminal leucocyte concentration did not correlate with SDF, unadjusted ORP, ORP normalised for sperm concentration (sORP), ORP normalised for total motile sperm concentration (motORP) or total motile sperm count (TMSC-ORP). Although receiver operator characteristic (ROC) curve analyses show that leucocytospermia does not predict high sORP values (>1.34 mV/10⁶ spermatozoa/ml), the motORP (AUC: 0.666) and TMSC-ORP (AUC: 0.683) predict the rate of leucocytospermia significantly (p = .0195 and p = .0085 respectively). Moreover, SDF can significantly predict leucocytospermia (AUC: 0.679; p = .011) and vice versa (AUC: 0.657, p = .0298). Our data confirm the association between OS and SDF. In conclusion, motORP and TMSC-ORP may be better predictive factors of leucocytospermia, probably because sperm motility, included in motORP and TMSC-ORP calculation, is the first seminal parameter to be affected by OS. Although all these parameters are indicative of OS, ORP values, SDF and leucocytospermia should be considered independently for the evaluation of redox seminal status, as they probe distinct seminal features.     

Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics

The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patient's biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty‐seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.     

Association of XRCC1 and ERCC2 promoters’ methylation with chromatin condensation and sperm DNA fragmentation in idiopathic oligoasthenoteratozoospermic men

The aim of the study was to investigate whether the promoter methylation of XRCC1 and ERCC2 genes is associated with sperm DNA fragmentation and chromatin condensation in idiopathic oligoasthenoteratozoospermic men. This study involved 77 infertile men with idiopathic oligoasthenoteratozoospermia and 51 normozoospermic controls. The methylight method, TUNEL assay and aniline blue staining were used for the evaluation of XRCC1 and ERCC2 genes’ methylation, SDF and sperm chromatin condensation, respectively. SDF (p = .004) and XRCC1 methylation (p = .0056) were found to be significantly higher in men with idiopathic OAT than in the controls, while mature spermatozoa frequency was higher in controls as compared to infertile men (p < .0001). No significant association was found between SDF and methylation of XRCC1 and ERCC2 genes (p = .9277 and p = .8257, respectively). However, compared to the cut-off point obtained by receiver operating characteristic analysis, a significant association was found between SDF and XRCC1 methylation, positive and negative methylation groups, generated according to the cut-off value for XRCC1. XRCC1 methylation was found to have a significant effect on chromatin condensation (p = .0017). No significant difference was detected among ERCC2 methylation, male infertility and SDF. In conclusion, XRCC1 methylation may have a role in sperm chromatin condensation and idiopathic OAT.     

The efficacy of antioxidants in sperm parameters and production of reactive oxygen species levels during the freeze-thaw process: A systematic review and meta-analysis

To investigate the impact of antioxidants in sperm parameters and reduction in reactive oxygen species production during the freeze-thaw process. PubMed, Scopus, Web of Science, Embase and Cochrane central library were systematically searched. Of the 1583 articles, 23 studies were selected for data extraction. Our results show that antioxidants improved sperm progressive motility (standardised mean difference (SMD) = 1; 95% CI: 0.62, 1.38; p < .001) and viability (SMD = 1.20; 95% CI: 0.50, 1.91; p = .001) and reduced sperm DNA fragmentation (SDF) and hydrogen peroxide (H₂O₂) production, but there was no significant improvement in total sperm motility after thawing. Acetyl-l-carnitine/l-carnitine, melatonin and catalase had a significant positive impact on progressive motility. The role of tempol and melatonin in improving viability was significant compared to other antioxidants. Moreover, a significant reduction in SDF was observed after addition of butylated hydroxytoluene, tempol and vitamin E. However, the prevention of H₂O₂ production was significant only after the addition of tempol. Our overall results displayed the positive impact of antioxidants on progressive sperm motility, viability and reduction in SDF and H₂O₂ production, but no significant impact of antioxidants on total sperm motility was seen during the freeze-thaw process.     

Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD‐HaloSpermG2®) comparable?

Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer‐based sperm chromatin structure analysis (SCSA) and the new light‐microscopy‐based sperm chromatin dispersion assay (SCD‐HaloSpermG2^®), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p < 0.05) higher in patients (DFI: 40.2% ± 3.0 vs. SDF: 40.3% ± 1.4) than in fertile donors (DFI: 17.1% ± 2.1 vs. SDF: 20.9% ± 2.5). Sperm selection led to lower proportions of DNA‐fragmented spermatozoa (DFI: 11.9 ± 1.7 vs. SCD: 10.0 ± 0.9, p < 0.05). The techniques output correlated highly and significantly (r² = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.     

A methodological validation of an easy one-step swimout semen preparation procedure for selecting DNA fragmentation-free spermatozoa for ICSI

The main purpose of this methodological paper was to describe a recently designed one-step ICSI semen preparation swim-out method (called swim-ICSI) and to compare its efficacy with our conventional two-step swim-out method for the selection of motile spermatozoa for ICSI with minimal DNA damage. In this observational cohort study, 42 fresh ejaculate sperm samples for ICSI were included to compare the new swim-ICSI with the conventional swim-out. In a sub-analysis (n = 20), both in-house designed ICSI preparation methods were compared with a commercial magnetic-activated cell sorting test (MACS^®). Sperm DNA fragmentation (SDF), using Halosperm^®, was determined at different time points during sperm preparation: on the native sample (a), after density gradient centrifugation (DG) (b), on the motile (A + B) spermatozoa selected with conventional swim-out post-DG (c) and selected with swim-ICSI method post-DG (d). For a subgroup (n = 20), SDF was also calculated after MACS (e). The mean SDF significantly reduced after EACH preparation step and reduced to almost zero in the recovered A + B spermatozoa when the semen prepared with DG was further processed for ICSI (swim-ICSI vs. swim-out, p = .001). In conclusion, the optimised one-step and fine-tuned swim-ICSI technique shows the possibility to select a population of spermatozoa with almost zero SDF to be used in ICSI treatments.     

The role of sperm in inducing genomic changes in the implantation: An experimental study

Endometrial receptivity and implantation are important topics in reproductive sciences. No evidence was found to support sperm involvement in endometrial receptivity and its associated factors. This study aimed to explore the effect of the normal human spermatozoa–endometrium cell interaction in regulating genes in the endometrial receptivity pathway. Semen samples were collected from a healthy and fertile man; then, they were incubated with endometrial cells for 24 hr and considered as the sperm group. A group was cultured without spermatozoa and considered as a control group. About 24 hr later, cells were collected from the bottom of the culture dish. The expressions of the VEGF, FGF2, HBEGF, LIFR, EGF, LIF, MUC1, HOXA10, CSF and PGR genes were evaluated in the two groups. Statistical analysis was performed using an independent sample test. Compared with the control group, in the sperm group, the mRNA levels of PGR (p = .0451), VEGF (p = .0101), HBEGF (p = .0163), EFG (p = .0339), FGF2 (p = .012), LIF (p = .0324), LIFR (p = .0321) and HOXA10 (p = .0098) were significantly upregulated. The results showed that there is a need for the interaction between spermatozoa and endometrium for implantation and can be used for preparing uterine in in vitro fertilisation cycles.     

The effect of cigarette smoking on human seminal parameters,sperm chromatin structure and condensation

Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11‐20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking.     

Do assisted reproduction outcomes differ according to aetiology of obstructive azoospermia?

Azoospermia is defined as absence of spermatozoa and may be secondary to blocked seminal ducts, known as obstructive azoospermia. Semen quality may be impaired due to factors such as sperm cell DNA fragmentation and presence of antisperm antibodies. The objective of this article was to investigate potential differences in outcomes of in vitro fertilisation and intracytoplasmic sperm injection between groups with different obstruction aetiology, as well as between the use of different techniques and sperm cells of different origins. Retrospective, multi-centre analysis of 621 first cycles was carried out between 2008 and 2015: Group I, congenital obstruction, 45 patients and Group 2, vasectomy, 576 patients. Sperm cell retrieval was achieved in all cases. Results were similar for Group I and II fertilisation rates, 70% versus 66.85% (p = .786); pregnancy rates, 42.5% versus 41.46% (p = .896); and live birth rates, 29.73% versus 17.69% (p = .071). According to sperm cell origin (579 epididymal vs. 42 testicular), pregnancy rates, 41.47% versus 43.9% (p = .760); and live birth rates, 18.3% versus 27.78% (p = .163) had no difference. Fertilisation, pregnancy and live birth rates did not differ according to obstruction aetiology. Outcomes did not differ between groups according to sperm cell origin.     

The progesterone‐induced sperm acrosome reaction is a good option for the prediction of fertilization in vitro compared with other sperm parameters

Progesterone (P₄) is crucial for the physiological function of spermatozoa. In the study, we investigated the correlation between P₄‐induced sperm acrosome reaction (AR) and parameters including sperm progressive motility, normal morphology and sperm DNA fragmentation (SDF), and compared the in vitro fertilization (IVF) predictive values of these indicators based on the multivariate regressions analysis and receiver operator characteristics (ROC) curve analyses. The results demonstrated a negative correlation between P₄‐induced sperm AR and the SDF, with the correlation ?9.05 (?17.25 to ?0.84), p<0.05, n = 47). No relationship was found between the sperm progressive motility, normal morphology and the induced AR. The P₄‐induced AR and SDF were both significantly correlated to the fertilization rate. ROC curve analyses indicated that P₄‐induced AR was a better prognostic predictor for the fertilization rate compared with the SDF, with the areas under the curve 0.729 (0.580–0.849), p<0.01 and 0.637 (0.484–0.772), p=0.16 respectively. The cut‐off value for P₄‐induced AR to predict “50% fertilization rate” was 23.4% with sensitivity and specificity of 63.3% and 88.2% respectively. The overall results indicated that the assessment of P₄‐induced AR seemed to be a more sensitive indicator for fertilization rate in vitro compared with other sperm parameters.     

In vitro effects of clomiphene citrate on sperm parameters and fertilisation rate in male NMRI mice

Clomiphene citrate (CC), as a medication in male infertility, improves the sperm parameters in oral consumption but various detrimental side effects have been reported including testicular tumours, gynaecomastia, skin allergic reactions and ocular symptoms. Therefore, this study was designed to evaluate the in vitro effects of CC on sperm parameters and fertilisation rate in IVF protocol. Sperm samples of NMRI adult mice were divided into six groups: group 1 received no treatment (control group), while groups of 2, 3, 4, 5 and 6 (experimental groups) were incubated with the doses of 0.001, 0.01, 0.1, 1 and 10 µg/ml of CC in culture medium respectively. Sperm parameters (viability, morphology and motility), DNA fragmentation levels and fertilisation rate in IVF were evaluated. The results demonstrated that the doses of 0.1 µg/ml (p = .000007 for viability and p = .00006 for fertilisation rate) and 1 µg/ml (p = .032 for viability and p = .005 for fertilisation rate) CC cause a significant improvements; also, the dose of 0.1 µg/ml CC found effective on sperm motility (p = .0003). In the field of IVF, the application of 0.1 and 1 µg/ml of CC in the culture medium may improve the sperm parameters in IVF protocol with no side effects.     

ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa

Sperm DNA fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential with contradictory results regarding its effect on ICSI. The aim of this study was to evaluate intracytoplasmic sperm injection (ICSI) outcomes in male patients with high SDF using testicular versus ejaculated spermatozoa. This is a prospective study on 36 men with high‐SDF levels who had a previous ICSI cycle from their ejaculates. A subsequent ICSI cycle was performed using spermatozoa retrieved through testicular sperm aspiration. Results of the prior ejaculate ICSI were compared with those of the TESA‐ICSI. The mean (SD) SDF level was 56.36% (15.3%). Overall, there was no difference in the fertilization rate and embryo grading using ejaculate and testicular spermatozoa (46.4% vs. 47.8%, 50.2% vs. 53.4% respectively). However, clinical pregnancy was significantly higher in TESA group compared to ejaculated group (38.89% [14 of 36] vs. 13.8% [five of 36]). Moreover, 17 live births were documented in TESA group, and only three live births were documented in ejaculate group (p < .0001). We concluded that the use of testicular spermatozoa for ICSI significantly increases clinical pregnancy rate as well as live‐birth rate in patients with high SDF.     

Supplementing post-wash asthenozoospermic human spermatozoa with coenzyme Q10 for 1 hr in vitro improves sperm motility,but not oxidative stress

We investigated the effect of supplementing post-wash asthenozoospermic spermatozoa with coenzyme Q10 (CoQ10) in vitro, which may reduce oxidative stress and improve sperm motility. Semen samples were collected from 39 men with asthenozoospermia, and their spermatozoa were isolated by two-layer Percoll density-gradient centrifugation. Kinetic parameters of the isolated spermatozoa (baseline before intervention) were determined immediately by computer-aided semen analysis. Total anti-oxidant capacity and protein carbonyl levels, as markers of oxidative stress, were also measured in the baseline spermatozoa. The baseline spermatozoa suspension was divided equally into two portions, one for CoQ10 supplementation (50 µg/ml for 1 hr) and the other as an un-supplemented vehicle control. The total motility of the CoQ10-supplemented spermatozoa was significantly higher than in the control (p = .009) and progressive motility tended to be higher (p = .053). Immotile sperm concentration in the CoQ10-supplemented spermatozoa was significantly lower than in both the baseline (p = .026) and control (p = .009). Total anti-oxidant capacity and protein carbonyl levels between the baseline, CoQ10-supplemented and control spermatozoa were not significantly different. Our data suggest that CoQ10 treatment reactivated sperm motility. We propose short-term supplementation of post-wash asthenozoospermic spermatozoa with CoQ10 before intrauterine insemination.     

Does sperm DNA fragmentation have negative impact on embryo morphology and morphokinetics in IVF programme?

Evaluation of sperm integrity may predict the in vitro fertilisation (IVF) outcomes. The aim was to evaluate the relationship between the sperm DNA fragmentation (sDNAf) with embryo morphology and morphokinetic using time-laps monitoring (TLM) and to select the best time points for normalisation in IVF setting. After evaluating the fertilisation and pronuclei (Z) scoring, 328 normally fertilised oocytes were assessed to time of pronuclei fading, time of 2 to 8 discrete cells (t2–t8) and abnormal cleavage patterns, such as multinucleation, direct cleavage, reverse cleavage and fragmentation. Sperm chromatin dispersion (SCD) assay was used for assessment of prepared sperm chromatin status. SCD was categorised into 4 groups of <6.5, 6.5–10.7, 10.7–20.1 and >20.1. The finding showed significant differences in t6 (p = .012), t7 (p = .045), t8 (p = .013) and s1 (p = .001) between 4 SCD groups. When morphokinetic variables were normalised to tPNf, this difference was observed in t2 (p = .003) and t6 (p = .017). Subsequently, the percentage of top quality embryos and Z₁ scoring were dependent to the sDNAf rate. In conclusion, tPNf was the best reference time point in IVF cycles. Also, we found high sDNAf rate had no negative impact on embryo morphology and morphokinetics in conventional IVF.     

The presence of seminal plasma,especially derived from stallion semen,helps preserve chilled Asian elephant (Elephas maximus) sperm motility

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris–glucose–egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.     

Famotidine does not affect human sperm fertility characteristics (motility,viability and DNA integrity) in vitro

Famotidine, a histamine‐2 receptor antagonist, is commonly used to relieve the acid‐related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty‐nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin–nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.     

Double‐blind,randomised, placebo‐controlled trial on the effect of L‐carnitine and L‐acetylcarnitine on sperm parameters in men with idiopathic oligoasthenozoospermia

Carnitine is essential for energy metabolism and spermatozoa maturation. Combining L‐carnitine and L‐acetylcarnitine with micronutrients has been investigated as a treatment for infertility in men. We evaluated the effects of a therapeutic formulation, Proxeed Plus, on sperm parameters in oligoasthenozoospermic men. This prospective, randomised, double‐blind, placebo‐controlled clinical trial involved 175 males (19–44 years) with idiopathic oligoasthenozoospermia who failed to impregnate their partners (12 months). Males received Proxeed Plus or placebo for 3 and 6 months. Sperm volume, progressive motility and vitality significantly (p < 0.001) improved after 6 months compared to baseline. Sperm DNA fragmentation index significantly decreased compared to baseline (p < 0.001) and the 3‐month therapy (p = 0.014) in treated men. Increased seminal carnitine and α‐glucosidase concentration also positively correlated with improved progressive motility. Decreased DNA fragmentation index was the good predictor of progressive sperm motility >10%, and simultaneous measurement of changes in sperm vitality and DNA fragmentation index gave the highest probability of sperm motility 10% (AUC = 0.924; 95% CI = 0.852–0.996; p < 0.001). Logistic regression analyses revealed DNA fragmentation index decrease as the only independent predictor of sperm motility 10% (OR = 1.106; p = 0.034). We have demonstrated the beneficial effects of carnitine derivatives on progressive motility, vitality and sperm DNA fragmentation. Combining metabolic and micronutritive factors is beneficial for male infertility.     

Genetic,biochemical and histopathological evaluations of thymoquinone on male reproduction system damaged by paclitaxel in Wistar rats

This study was aimed to evaluate therapeutic effects of thymoquinone on male reproductive damages induced by paclitaxel. Forty-eight male rats were divided; control, paclitaxel (4 mg/kg), paclitaxel + thymoquinone (1.25, 2.5 and 5 mg/kg) and thymoquinone (1.25, 2.5 and 5 mg/kg). Paclitaxel and thymoquinone were administrated intraperitoneally for 4 and 14 days respectively. Then, the testes were removed for H&E staining, sperm parameters and apoptotic genes expression assessments. Serum levels of nitric oxide, total antioxidant capacity and testosterone were evaluated, and sperm DNA fragmentation was assessed. Paclitaxel significantly (p < .05) increased nitric oxide, decreased total antioxidant capacity and reduced testosterone levels than control group. Sperm motility, viability and count were significantly (p < .05) reduced in paclitaxel group than control. Co-administration of thymoquinone + paclitaxel caused decreased levels of nitric oxide and increased total antioxidant capacity, testosterone levels and reproductive parameters than paclitaxel group significantly (p < .05). Paclitaxel significantly (p < .05) increased caspase-3 and p-53 and decreased Bcl-2 genes expression than control. Sperm DNA fragmentation index was also increased significantly (p < .05) in paclitaxel group than control, and this value was decreased in whole doses of paclitaxel + thymoquinone groups than paclitaxel. Thymoquinone can alleviate the side effects of paclitaxel on the male reproductive system.