Optimization of the microbiological treatment of sperm for assisted reproductive technology (ART).

In ART,1 optimized sperm preparation could separate spermatozoa from potentially harmful germs and cells, improving sperm parameters. This study compares the outcome of a routinely used two-layer density gradient with a three-layer protocol in terms of sperm and microbiological parameters. According to the results, a three-layer protocol was more effective.

Sperm Preparation with Microfluidic Sperm Sorting Chip May Improve Intracytoplasmic Sperm Injection Outcomes Compared to Density Gradient Centrifugation.

Does sperm preparation using the FERTILE PLUS™ Sperm Sorting Chip improve fertilization rates, blastocyst formation, utilization, and euploidy rates in patients undergoing intracytoplasmic sperm injection (ICSI), compared with density gradient centrifugation (DGC)? A single-cohort, retrospective data review including data from 53 couples who underwent ICSI cycles within a 12-month period. For each couple, the two closest, consecutive cycles were identified, where one used the standard technique of sperm preparation (DGC) and the subsequent used FERTILE PLUS™, therefore, couples acted as their own controls. Paired samples t-test was used to compare means for the outcomes (fertilization, blastocyst formation, utilization, and euploidy rates). Binary logistic regression analysis assessed the relationship between female age, the presence of male factor infertility, and euploidy rates. Blastocyst, utilization, and euploidy rates were significantly higher for cycles using FERTILE PLUS™ compared to DGC (76% vs 56%, p = 0.002; 60% vs 41%, p = 0.005, and 40% vs 20%, p = 0.001, respectively). Although there was an increase in fertilization rates for cycles using FERTILE PLUS™, this was not significant (72% vs 68%, p = 0.449). The euploidy rates of females ≤ 35 years were significantly increased when the FERTILE PLUS™ sperm preparation method was used, compared to the older age group (OR 2.31, p = 0.007). No significant association was found between the presence or absence of male factor infertility and euploidy rates between the two cycles. This study provides tentative evidence that the FERTILE PLUS™ microfluidic sorting device for sperm selection can improve blastocyst formation, utilization, and euploidy rates following ICSI in comparison to the DGC method.

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting.

PURPOSE: A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. METHODS: Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60 min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). RESULTS: Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30 min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CONCLUSION: CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

The impact of different sperm preparation methods on clinical pregnancy and live birth rates in intrauterine insemination cycles: a retrospective single-center cohort study.

OBJECTIVE: To assess the impact of 2 different sperm preparation methods, density gradient centrifugation and simple wash, on clinical pregnancy and live birth rates in intrauterine insemination (IUI) cycles with and without ovulation induction. DESIGN: Retrospective single-center cohort study. SETTING: Academic fertility center. PATIENTS: In total, 1,503 women of all diagnoses sought IUI with fresh-ejaculated sperm. EXPOSURE: Cycles were divided into 2 groups on the basis of sperm preparation technique: density gradient centrifugation (n = 1,687, unexposed group) and simple wash (n = 1,691, exposed group). MAIN OUTCOME MEASURES: Primary outcome measures consisted of clinical pregnancy and live birth rates. Furthermore, adjusted odds ratios and 95% confidence intervals for each outcome were calculated and compared between the 2 sperm preparation groups. RESULTS: Odds ratios did not differ between density gradient centrifugation and simple wash groups for clinical pregnancy and live birth (1.10 [0.67-1.83] and 1.08 [0.85-1.37], respectively). Additionally, when cycles were stratified using ovulation induction rather than adjusted for, no differences in clinical pregnancy and live birth odds were noted between sperm preparation groups (gonadotropins: 0.93 [0.49-1.77] and 1.03 [0.75-1.41]; oral agents: 1.78 [0.68-4.61] and 1.05 [0.72-1.53]; unassisted: 0.08 [0.001-6.84] and 2.52 [0.63-10.00], respectively). Furthermore, no difference was seen in clinical pregnancy or live birth when cycles were stratified using sperm score or when the analysis was limited to first cycles only. CONCLUSION: Overall, no difference was noted in clinical pregnancy or live birth rates between patients who received simple wash vs. density gradient-prepared sperm, suggesting similar clinical efficacy between the 2 techniques for IUI. Because the simple wash technique is more time-efficient and cost-effective compared with the density gradient, adoption of this technique could lead to comparable clinical pregnancy and live birth rates for IUI cycles, although optimizing teamwork flow and coordination of care.

A review of the use of antioxidants in bovine sperm preparation protocols.

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function.

Spermatozoa isolation with Felix™ outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage.

BACKGROUND: The optimization of spermatozoa preparation techniques in order to obtain cell fractions enriched with structurally and functionally "superior" spermatozoa is a key objective of the assisted reproduction industry. OBJECTIVES: The purpose of this study was to evaluate a recent development of an electrophoretic spermatozoa separation device (Felix™, Memphasys Ltd, Sydney, Australia) and to compare its performance with conventional spermatozoa preparation by density gradient centrifugation (DGC). Particular attention was paid to the evaluation of sperm DNA/nuclear integrity. MATERIALS & METHODS: A cohort of 29 human semen samples was studied. Semen samples were analyzed fresh and after DGC or Felix™ preparation. Semen parameters monitored included sample volume, sperm count, total motility, progressive motility, sperm DNA fragmentation using the Sperm Chromatin Structure Assay and sperm DNA oxidation. RESULTS: Spermatozoa preparation with Felix™ resulted in significantly improved spermatozoa fractions with higher progressive motility, lower sperm DNA fragmentation, and lower sperm DNA oxidation compared with raw semen and DGC-prepared spermatozoa. DISCUSSION & CONCLUSION: The data collected in this study support the preparation of spermatozoa by the Felix™ system as it allows selection of spermatozoa with the highest progressive motility as well as the lowest nuclear/DNA damage. These improved sperm parameters, along with the fact that the Felix™ separation process is very fast and highly standardized, should be of great interest to the assisted reproduction technologies industry.

Evaluation for optimization effects of sperm floating plate / 临床检验杂志

Objective To compare the difference between sperm floating plate and density gradient centrifugation combined with swim-up in human sperm preparation.Methods The semen samples were obtained from 50 infertile men in the clinic of Reproductive Medi-cine of Northwest Women's and Children's Hospital excluding azoospermia,severe oligoasthenozoospermia and semen volume less than 2 mL.After semen liquefaction,the differences of sperm concentration,total progressively motile sperm count(TMSC),percentages of progressively motile sperm and normal morphology sperm,recovery rate and DNA fragmentation index(DFI)were measured by both the methods of sperm floating plate and density gradient centrifugation combined with swim-up,and the results were compared.Results Compared with the pre-sorting samples,sperm concentrations[(16.08±13.39)x 106/mL,(8.88±8.06)x 106/mL vs(60.05± 27.21)×106/mL],TMSC[(7.41±6.14)×106,(3.98±3.57)×106vs(22.24±13.74)×106]and DFI[(2.20±3.44)％,(5.20± 10.79)％vs(26.38±13.92)％]in the sorting groups by sperm floating plate and density gradient centrifugation combined with swim-up were decreased significantly,and the percentages of progressive motile sperm[(91.67±4.75)％,(87.86±7.90)％vs(40.21± 16.83)％]and normal morphology sperm[(9.58±5.08)％,(7.72±4.01)％vs(3.58±2.06)％]were increased significantly.Com-pared with the density gradient centrifugation combined with swim-up,the results of sperm floating plate were higher in sperm concen-tration,percentages of progressively motile sperm and normal morphology sperm,TMSC and sperm recovery rate[(30.74±13.70)％vs(17.09±9.20)％],but DFI was lower,time-consuming was shorter[(32.38±1.01)min vs(60.08±2.06)min],and the difference between the two groups was statistically significant(P＜0.05).Conclusion The sperm floating plate may have certain clinical applica-tion prospects in the future due to better parameters of sperm preparation than those of density gradient centrifugation combined with swim-up in simple operation and shorter time-consuming.

Free centrifuge sorting method for high-count sperm preparation improves biological characteristics of human spermatozoa and clinical outcome: A sibling oocytes study.

Sperm processing for assisted reproductive technologies (ART) aims to separate immotile and debris from the motile spermatozoa in the semen. The purpose of this study was to assess the effect of free centrifuge sorting (FCS) approach based on a combination of rheotaxis and swim-up on sperm biological characteristics and ICSI clinical outcomes. Each semen sample was splitted into two equal parts for 67 ICSI cycles with donation oocytes. Parts were processed with the Direct Swim Up (DSU) (control) and with the FCS method (experimental). Sperm quality was assessed in terms of motility, fine morphology, mitochondrial membrane potential, lipid peroxidation and sperm DNA fragmentation. Also Following ICSI, the clinical outcomes were compared between the groups. Sperm progressive motility (93.5 ± 4.1% vs. 78.6 ± 8.2%; p < 0.001), the fraction of Class I (good) morphology (30.2 ± 9.4% vs. 23.7 ± 8.5%; p < 0.0001) and the rate of mitochondrial membrane potential (77.4 ± 7.2% vs. 66.9 ± 5.7%; p < 0.0001) were significantly higher in the FCS compared to DSU groups. The level of lipid peroxidation (0.5 ± 0.05% vs. 0.6 ± 0.06%; p < 0.0001) and concentration of DNA fragmentation (DF) (7.4 ± 1.6% vs. 15.4 ± 2.6%; p < 0.0001) were lower in sperm from the FCS group compared to DSU group. There were higher rates of high-quality embryo formation (p < 0.001), implantation and clinical pregnancy rates (p = 0.03) in the FCS group compared to the control group. The processing of seminal samples using FCS collected spermatozoa with better biological quality and resulted in higher reproductive outcomes in ICSI cycles.

The Effectiveness of Sperm Preparation Using Density Mini-Gradient and Single-Layer Centrifugation for Oligospermia Samples.

Background: Sperm preparation is an important step during assisted reproduction, and different assisted reproductive techniques have different sperm quality requirements. For intrauterine insemination (IUI), the total motile sperm count is a predictor of a patient's fertility. Objective: The aim of this study was to compare the sperm recovery rate and DNA fragmentation index (DFI) outcomes following density mini-gradient and single-layer centrifugation in preparation for intrauterine insemination (IUI). Methods: A total of 30 semen samples with concentrations under 15 million cells/ml were obtained, and each sample was divided into 3 aliquots, with each aliquot subjected to 1 of 3 separation methods: mini-gradient, single-layer using a 90% density layer (single 90-layer), and single-layer using a 45% density layer (single 45-layer). Total sperm motility and sperm recovery rates were compared before and after preparation using each method. Results: The sperm concentration obtained using single 45-layer was higher than the other groups (p<0.05), but sperm motility was higher using the mini-gradient and single 90-layer methods higher than the single 45-layer method (p<0.05). The recovered sperm motility rates for the mini-gradient, single 90-layer, and single 45-layer methods were 57.6% ± 20.6%, 62.8% ± 18.5%, and 78.7% ± 12.4%, respectively, indicating a better outcome for the single 45-layer method than for the other methods. Conclusion: All of these methods can be applied to sperm preparation for IUI, and the optimal method can be selected based on initial sperm quality to collect sperm with good motility and DNA integrity to achieve a satisfactory pregnancy rate.

Comparison of sperm preparation methods to improve the recovery of mature spermatozoa in sub-fertile males.

The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

Effects of sperm preparation techniques on sperm survivability and DNA fragmentation.

OBJECTIVE: This study was performed to determine the effect of swim-up (SU) and density gradient centrifugation (DGC) on sperm survival and DNA fragmentation. METHODS: Individual semen samples were analyzed before each was divided into two aliquots (half for SU and half for DGC) for calculation of sperm survival and the DNA fragmentation index (DFI). Sperm DNA fragmentation was determined using the sperm chromatin dispersion test. RESULTS: The DFI of the 63 semen samples processed using both procedures was lower than that of the fresh semen samples. The DFI was significantly lower for samples processed using the SU than DGC method. In the sperm survival test, the SU technique was associated with increased sperm motility and vitality following preparation. After 24 hours, however, the concentration and percentage of surviving sperm were significantly lower in the SU than DGC group. CONCLUSIONS: Both semen preparation techniques help to minimize sperm DNA fragmentation; however, when the DFI is <30%, the SU technique is more appropriate than DGC. While DGC may be superior for intrauterine insemination, the SU method may be preferable for in vitro fertilization or maturation.

Cumulative live birth rates after IVF/ICSI cycles with sperm prepared by density gradient centrifugation vs. swim-up: a retrospective study using a propensity score-matching analysis.

BACKGROUND: Density gradient centrifugation (DGC) and swim-up (SU) are the two most widely used sperm preparation methods for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, existing comparisons of IVF/ICSI outcomes following these sperm preparation methods are insufficient and controversial. METHODS: This retrospective study included all first autologous IVF and ICSI cycles performed between March 1, 2016, and December 31, 2020 in a single university-based center. A total of 3608 cycles were matched between DGC and SU using propensity score (PS) matching for potential confounding factors at a ratio of 1:1. The primary outcome was the cumulative live birth rate (cLBR) per aspiration. RESULTS: PS matching provided 719 cycles after DGC and 719 cycles after SU. After adjusting for confounders, the recovery rate, progressive motility rate after sperm preparation, fertilization rate, good-quality embryo rate, and blastocyst formation rate were similar between the DGC and SU groups. The cLBR (odds ratio [OR] = 1.143, 95% confidence interval [CI]: 0.893-1.461) and LBR per transfer (OR = 1.082, 95% CI: 0.896-1.307) were also not significantly different between the groups. Furthermore, no significant differences were found in all of the laboratory and clinical outcomes following conventional IVF or ICSI cycles between the two groups. However, a significantly higher fertilization rate (ß = 0.074, 95% CI: 0.008-0.140) was observed when using poor-quality sperm in the DGC group than in the SU group. CONCLUSIONS: Sperm preparation using DGC and SU separately resulted in similar IVF/ICSI outcomes. Further studies are warranted to compare the effects of these methods on IVF/ICSI outcomes when using sperm from subgroups of different quality.

A Simple, Centrifugation-Free, Sperm-Sorting Device Eliminates the Risks of Centrifugation in the Swim-Up Method While Maintaining Functional Competence and DNA Integrity of Selected Spermatozoa.

This pilot study was conducted to explore the benefits of using a centrifugation-free device based on the migration-sedimentation (MS) technique over centrifugation-based techniques in selecting competent spermatozoa, as compared with using split human semen samples. Ejaculates from 35 men undergoing semen analysis were split into four parts where one part was retained as the neat (NE) and the other three parts were subjected to sperm selection by using migration-sedimentation (MS), density gradient (DG) separation, and swim-up (SU) techniques. Sperm functional characteristics along with mitochondrial integrity, tyrosine phosphorylation, acrosome reaction, and ultrastructure were measured. The ability of selection techniques in reducing spontaneous and radiation-induced sperm DNA lesions was assessed by the TUNEL assay. In results, MS-selected spermatozoa had higher viability (P < 0.001), longevity in terms of total motility at the end of 6 and 18 h post-extraction (P < 0.001), and mitochondrial integrity (P < 0.001) compared with those selected by DG. Furthermore, spontaneous DNA lesions were significantly reduced in MS and SU fractions compared with NE (P < 0.001). Similarly, radiation-induced sperm DNA lesions were significantly lower in MS and SU fractions (P < 0.001) compared with DG. Ultrastructural analysis using scanning electron microscopy suggested a moderate, non-significant increase in the number of spermatozoa with normal head and mid-piece in MS fraction compared with other methods. In conclusion, the MS-based device offers a centrifugation-free, efficient, and reliable sperm selection method, making it suitable for partially equipped intra-uterine insemination (IUI) laboratories or office IUI programmes. Further research should focus on the safety and clinical usefulness of the device in assisted conception programmes in general and IUI in specific.

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.

Hypotonic challenge reduces human sperm motility through coiling and folding of the tail.

Human ejaculates collected for in vitro procedures show variably rapid increases in osmolality, depending on enzymatic degradation of compounds. Changes in osmolality can affect cell functions due to the energy consuming processes needed to control cell volume. The aim was to examine the effects of a hypotonic challenge for spermatozoa exposed to increased osmolality. Spermatozoa were selected by density gradient centrifugation and washed in media with different osmolalities. Osmolality was measured by freezing-point depression and sperm velocities by CASA. Swimming pattern observations and assessments of tail morphology of fixed spermatozoa were done with phase contrast microscopy. Increased osmolality did not change the curvilinear velocity (VCL), while decreased osmolality reduced or abolished VCL nonreversibly. For spermatozoa first exposed to 400 mOsm/kg, reversal of osmolality to 290 mOsm/kg reduced the VCL and the average path velocity (VAP) permanently. Hypotonic challenges increased sperm tail coiling and folding in a dose-response pattern. Spermatozoa once adjusted to high osmolality in the liquefied ejaculate are likely to suffer if exposed to a medium with a lower osmolality. For improved success of Assisted Reproductive Technologies (ART), it appears to be important to minimise the duration of sperm exposure to the ejaculate, by early dilution or sperm preparation.

A fast and safe technique for sperm preparation in ICSI treatments within a randomized controlled trial (RCT).

Recently a novel method based on horizontal sperm migration in injection dishes has been introduced as an additional tool for preparation of semen sample in assisted reproductive technology (ART) procedures. In the present study, we evaluated both timing and reproductive outcomes in a randomized controlled study including 1034 intra-cytoplasmic sperm injection (ICSI) procedures followed by fresh embryo transfer. Couples enrolled were divided into two sub-groups, namely conventional swim-up method (Group A), and horizontal sperm migration in injection dishes (Group B).No significant differences were found between groups with respect to fertilization rate, implantation success, clinical pregnancy outcomes and ongoing pregnancies. On the contrary, both cleavage and blastocyst rates were statistically higher in Group B, suggesting superior efficiency and safety of this innovative technique also including time-saving and cheaper costs as compared to the classical swim-up sperm preparation.Our data support the interpretation of the horizontal sperm migration as a promising procedure for semen preparation in ART cycles.

New device for sperm preparation involving migration-gravity sedimentation without centrifugation compared with density-gradient centrifugation for normozoospermic intrauterine insemination.

OBJECTIVE: To investigate the efficacy of a new device for sperm preparation involving migration-gravity sedimentation without centrifugation (MIGLIS), compared with density-gradient centrifugation (DGC) for normozoospermic intrauterine insemination (IUI). DESIGN: Retrospective cohort study. SETTING: Not applicable. PATIENTS: A total of 10,318 cases of IUI (3,015 MIGLIS and 7,303 DGC) between October 2013 and September 2019. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Sperm analysis, subsequent pregnancy outcomes, and complications. RESULTS: MIGLIS was associated with a lower sperm recovery rate and fewer injected sperm compared with DGC. However, the overall pregnancy rates following MIGLIS and DGC were similar (MIGLIS 8.8%, DGC 9.3%). In a subanalysis according to age, the pregnancy rate was higher for MIGLIS among women 40-41 years of age (8.6% vs. 5.9%). Peritonitis was the only recorded complication, with similar frequencies in the MIGLIS and DGC groups (MIGLIS two cases, DGC four cases). No cases became severe, and all improved after antibiotic treatment. There were no cases of uterine cramping or pain symptoms. CONCLUSIONS: MIGLIS is a new sperm preparation method that does not require centrifugation. Its use was associated with pregnancy rates similar to those with DGC and a higher pregnancy rate in older women. MIGLIS is a novel sperm preparation method for selecting spermatozoa with high motility and good fertilization ability in patients undergoing IUI, in vitro fertilization, and intracytoplasmic sperm injection.

Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate.

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double-strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double-strand breaks (r = -0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.