Sperm preparation is a technique used to minimize the factors that may affect sperm quality and reduce fertilization ability ^[1]. Human semen consists of several components, such as normal and abnormal spermatozoa, leukocytes, epithelial cells, and debris, as well as necrotic and immature content that form reactive oxygen species (ROS)^[2]. Physiologically, ROS plays an important role in maintaining sperm capacitation and the acrosome reaction ^[3]. On the other hand, the elevated level of ROS negatively affects the spermatozoa by inducing lipid peroxidation within the plasma membrane, leading to attenuation in sperm motility and alteration in morphology, in addition to decondensation and fragmentation in sperm DNA. The final result is the impaired fertilization potential of spermatozoa ^[4].

The sperm preparation techniques came to help select healthy motile spermatozoa that are morphologically normal and have intact DNA for use in assisted reproduction technologies (ART) procedures. The principles of the preparation techniques are based on migration, filtration, or density gradient centrifugation ^[5]. The common sperm preparation technique used in most ART laboratories is the swimming-up technique, which is carried out by adding the preparation media above the semen (direct swim-up) or above the pellet (pellet swim-up), and the motile spermatozoa migrate upward into the preparation media [6,7]. Despite being widely adopted, this technique has more than one disadvantage related to the centrifugation steps and continued cell-to-cell attachment in the pellet, resulting in increased generation of ROS [2,8,9]. Several studies demonstrate that centrifugation steps increase ROS generation and subsequently decrease sperm motility, and increase sperm DNA fragmentation (SDF) ^[1,10-12].

Because of the safety of the sperm is important for the in vitro fertilization (IVF) procedure, the current study aimed to use a centrifugation-free modified technique for selecting motile spermatozoa with intact DNA. 

Subjects and sample collection

The current study was performed on 70 semen samples from participants who attended the fertility center in Najaf City, Iraq, for semen analysis from February 2023 to July 2024. Samples with at least 1.5 ml volume, a concentration of ≥ 15 million sperm/ml, and progressive motility of ≥32% were incorporated in this study. Samples were taken from the participants on-site by masturbation after sexual abstinence for at least 3 days. Semen analysis was done according to the WHO laboratory manual to examine and process human semen (2010) ^[13]. After that, each sample was divided into three portions (P1-P3); each portion was prepared using one of the studied techniques: P1 swimming-up (SU), P2 pellet swimming-up (P-SU), and P3, the modified technique called “swimming-within” (S-Within). Sperm concentration, progressive motility, and morphology were reassessed after preparation. Furthermore, acrosome reaction (AR), mitochondrial activity (MA), and sperm DNA fragmentation (SDF) testing were conducted pre- and post-processing. Each parameter was assessed twice by a blinded, highly experienced technician to avoid observer bias. 

Ethical approval

The local medical ethics committee approved this study (Approval No: 23-6390), and all the patients signed an informed research agreement before providing the semen samples. 

Seminal fluid analysis

Depending on the WHO standard criteria (2010) 13, seminal fluid analysis for all samples was done within 1 hour of sample collection. The sperm parameters were determined using a light microscope (Optica, Italy). 

Sperm preparation techniques

1- Semen swimming-up (S-SU)

A volume of culture media (FertiCultTM Flushing medium, FertiPro, Belgium) was added over the same volume of the semen sample in a 14 ml sterile conical tube. The tube then leaned at an angle of 45° and incubated at 37°C and 6% CO₂  for 1 hour. Spermatozoa obtained from the top layer were utilized to evaluate the studied parameters after incubation ^[14].

2- Pellet swimming-up (P-SU) technique

In a sterile 14 ml conical tube, one volume of semen was diluted with culture media (FertiCultTM Flushing medium, FertiPro, Belgium) in a 1:2 ratio. After mixing, the mixture was centrifuged at 1500 rpm for 10 minutes; then, the supernatant was excluded, and 1ml of culture media (FertiCultTM Flushing medium, FertiPro, Belgium) was added again to cover the pellet. The tube was then laid at an angle of 45° and incubated at 37°C and 6% CO₂. for 1 hour. Spermatozoa obtained from the top layer (supernatant) were utilized to evaluate the studied parameters after incubation ^[14].

3- Swimming-within 

A 4mm kink-resistant pipe (4 cm in length) was used in this technique. One pipe end was cut at a 45° angle, and the other was connected to a medical syringe. 1 ml of the culture media (FertiCultTM Flushing medium, FertiPro, Belgium) was inserted into the pipe. A portion of the semen was laid within a 14 ml sterile tube, and the pipe was inserted into the tube. The tube was then leaned at an angle of 45° and incubated at 37°C and 6% CO₂ for 1 hour. The active spermatozoa swim into the nutrient-rich pipe. Following incubation, the pipe was removed from the tube, and the spermatozoa within the pipe were assessed for the studied parameters.

Assessment of sperm DNA fragmentation

Acridine Orange (AO) stain and fluorescent microscope technique were used to assess the SDF pre- and post-processing. In this technique, The AO stain, Tyrode’s solution, and the fixative solution (Carnoy’s solution) were prepared as provided [15]. 10 μL of the sample was drawn up on a glass slide and left to air dry. It was immersed within a freshly prepared Carnoy's solution and left overnight. The slides were then washed using distilled water and immersed in AO stain for 5 min. then, it was washed again and stored in a dark place until the evaluation day. A fluorescent microscope (Optica, Italy) was used to determine the SDF by measuring at least 200 spermatozoa at a magnification of 40X. the spermatozoa with intact DNA fluoresced green, while red or orange fluorescent appears with fragmented DNA[15,16]

Assessment of Acrosome Reaction

The acrosome reaction was evaluated using the triple-stain technique. In this technique, a portion of the sample was diluted with trypan blue stain 2% in a 1:1 ratio and underwent incubation at 37°C for 15 minutes. following incubation, 10 μL of the suspension was placed on a transparent microscope slide and immersed in glutaraldehyde 3% for 30 minutes to be fixed. Bismarck brown (0.8%) then was added for 10 minutes at 40°C as a counterstain. After that, Rose Bengal stain 0.8% was used for 45 minutes at 24°C for staining the acrosome. Under a light microscope, at least 200 spermatozoa were measured at a magnification of 100X. The viable spermatozoan appears brown, the dead spermatozoan appears blue, the intact acrosome appears pink, and the reacted acrosome appears white [17,18] 

Assessment of sperm mitochondrial Activity 

The technique used to assess the sperm mitochondrial activity relies on the oxidation, polymerization, and deposition of 3,3′-diaminobenzidine (DAB) by mitochondrial cytochrome c-oxidase, as described ^[19,20]. Briefly, a portion of the semen sample was mixed volume to volume with a solution containing 1 mg/mL of 3-3′-diaminobenzidine (DAB) in phosphate-buffered saline (PBS) in which (KCl 2.7 mM, NaCl 137 mM, Na₂HPO₄ 4.3 mM, and KH₂PO₄ 1.4 mM with pH of 7.4). The mixture was incubated in the dark for 1 hour at 37°C. then, 10 μL smears were placed on a microscope slide, and left to dry. After that, the slide was immersed in a fixative solution (10% formaldehyde) for 10 minutes and then rinsed and allowed to air dry again.

A differential interference contrast (DIC) microscope was used in this technique by measuring at least 200 spermatozoa under a magnification of 1000x. Based on the staining of their midpiece, the spermatozoa were classified into four distinct classes. Class I represents fully functional mitochondria, which exhibited complete staining in the midpiece. Class II showed staining in more than 50% of the midpiece, which indicates a minor decrease in mitochondrial activity that does not have a significant impact on their motility and fertilization ability. Class III had staining in less than 50% of the midpiece, which indicates that spermatozoa display notable mitochondrial malfunction, which prevents them from achieving hypermotility during capacitation. Class IV had no staining in the midpiece, these spermatozoa are either nonviable or have only limited energy produced by glycolysis.

Statistical analysis. 

The analysis of data was done using the software Statistical Package for the Social Sciences (SPSS) version 23 (IBM SPSS 23) for Mac. The parameters were expressed as mean ± standard deviation (SD). The analysis of variation (ANOVA) with the least significant difference (LSD) was used to compare the parameters. The p-value less than 0.01 was considered significantly different. 

Regarding sperm concentration, a significant decrease was seen in SU, P-SU, and S-within compared to raw semen (P < 0.01). The lowest sperm concentration was found in S-within (Table 1). The progressive motility (%) was significantly increased in SU, P-SU, and S-within compared to raw semen (P < 0.01). Interestingly, the highest level of progressive motility was in S-within, and it had a significant difference than SU and P-SU (P < 0.01). The normal morphology (%) was also significantly higher in spermatozoa prepared by S-within when compared with SU, P-SU, and row semen (P < 0.01) (Table 1).

Table 1: Comparison of semen basic parameters in the studied preparation techniques

The SU, P-SU, and S-within techniques recorded a significant decrease (P < 0.01) in SDF in comparison to the raw semen. Lower DNA fragmentation (P < 0.01) was detected in spermatozoa prepared by S-within rather than in the SU and P-SU techniques. In contrast, spermatozoa prepared by P-SU show a significantly higher DNA fragmentation than in SU and S-within (P < 0.01) (Figure 1A).

The percentage of spermatozoa with unreacted acrosome was significantly increased in all preparation techniques in comparison to raw semen (P < 0.01). The higher level was in S-within, and the lower level was in P-SU (Figure 1B) (P < 0.01).

Spermatozoa with class I mitochondria detected a significant increase in SU, P-SU, and S-within when compared to raw semen (P < 0.01) (Figure 2A). In contrast, spermatozoa with class II and class III were decreased significantly in P-SU and S-within compared with raw semen. Moreover, no significant difference was found in class III mitochondria between SU and raw semen. Interestingly, the higher class I mitochondria and the lower Class III and IV mitochondria levels were observed in spermatozoa prepared with the S-within technique (P < 0.01) (Figure 2A, C, and D).

Figure 1: (A) Sperm DNA fragmentation (%) in the studied preparation techniques, (B) Acrosome reaction (%) in the studied preparation techniques. Different letters (a, b, c, and d) on the bars indicate significant differences at p<0.01.

Figure 2: (A) Mitochondrial activity class I (%), (B) Mitochondrial activity class II, (C) Mitochondrial activity class III, and (D) Mitochondrial activity class IV in the studied preparation techniques. Different letters (a, b, c, and d) on the bars indicate significant differences at p<0.01.

The main purpose of preparation techniques is to select healthy motile spermatozoa from semen that contains immotile, morphologically abnormal, apoptotic, and dead spermatozoa ^[21]. Therefore, the preparation techniques can help select the highest concentration of healthy spermatozoa from raw semen [8]. 

Several preparation techniques are used in ART laboratories [2,22–28], but the effect of these techniques on sperm quality varies depending on the procedure's steps ²³. In this study, a modified simple procedure was used to minimize the negative effects of some steps in other procedures and increase the quality of prepared spermatozoa.

Seminal fluid contains sufficient antioxidants (ROS scavengers) that reduce the harmful effects of high ROS [29]. The ROS plays a significant physiological role in maintaining sperm capacitation and the acrosome reaction ^[3]. However, in preparation techniques, movement of spermatozoa from seminal fluid toward the overlaid preparation medium may induce an increase in the level of intracellular ROS, creating a disequilibrium between the level of ROS and the antioxidant capacity. This imbalance results in lipid peroxidation, loss or decrease in sperm motility, and increased DNA fragmentation [30,31]. ROS levels may increase during the centrifugation steps in many preparation techniques ^[32]. For instance, during the P-SU technique, semen centrifugation may accumulate abnormal and immotile spermatozoa, leukocytes, and debris in the pellet [24]. This increased cell-to-cell contact may impede the migration of motile spermatozoa toward the capacitating medium and increase the level of ROS ^[33]. This finding is in concordance with the results of the current study that found a high level of SDF in spermatozoa prepared by P-SU compared to SU and S-within. However, several studies found that using centrifugation with low force and minimum time can minimize the formation of high ROS levels, attracting healthy spermatozoa to the overlaid preparation medium [4,25,34]. Moreover, multi-step centrifugation in the density gradient technique leads to excessive ROS formation ^[7,35].

In the current study, a centrifugation-free technique was used to minimize the formation of ROS. Therefore, healthy spermatozoa can gravitate toward the preparation medium within the pipe. The pipe used, 4mm in diameter, may mimic the physiological environment, such as a fallopian tube. The new modified technique was compared with the most common techniques, SU and P-SU, and the results detected the superiority of the new technique (S-within) in selecting the high concentrations of motile spermatozoa with intact DNA and unreacted acrosome. 

The SU technique is also a centrifugation-free technique, and it was found “the best option for selecting healthier motile spermatozoa,” depending on the conclusion of [24] Interestingly, the present study demonstrates a significant increase in progressive motility, normal morphology, acrosome integrity, and class I mitochondrial activity in S-within compared to the SU technique. Furthermore, SDF was significantly decreased in S-within than in SU. 

 This superiority of the S-within technique may be due to the presence of the capacitating medium within the pipe and only the healthy motile spermatozoa swimming into the pipe, while in the SU technique, the capacitating medium loaded above the semen with the presence of chance to interfere with the semen and migrate of non-preferable spermatozoa to capacitating medium.

In conclusion, the S-within technique may be a good option for selecting a high concentration of motile spermatozoa with normal morphology, intact DNA, non-reacted acrosome, and high mitochondrial activity.

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the College of Medical Technology, The Islamic University, Najaf, Iraq.

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